Model-Based Drug-Drug Interaction Extrapolation Strategy From Adults to Children: Risdiplam in Pediatric Patients With Spinal Muscular Atrophy.

Risdiplam (Evrysdi) improves motor neuron function in patients with spinal muscular atrophy (SMA) and has been approved for the treatment of patients ≥2 months old. Risdiplam exhibits time-dependent inhibition of cytochrome P450 (CYP) 3A in vitro. While many pediatric patients receive risdiplam, a drug-drug interaction (DDI) study in pediatric patients with SMA was not feasible. Therefore, a novel physiologically-based pharmacokinetic (PBPK) model-based strategy was proposed to extrapolate DDI risk from healthy adults to children with SMA in an iterative manner. A clinical DDI study was performed in healthy adults at relevant risdiplam exposures observed in children. Risdiplam caused an 1.11-fold increase in the ratio of midazolam area under the curve with and without risdiplam (AUCR)), suggesting an 18-fold lower in vivo CYP3A inactivation constant compared with the in vitro value. A pediatric PBPK model for risdiplam was validated with independent data and combined with a validated midazolam pediatric PBPK model to extrapolate DDI from adults to pediatric patients with SMA. The impact of selected intestinal and hepatic CYP3A ontogenies on the DDI susceptibility in children relative to adults was investigated. The PBPK analysis suggests that primary CYP3A inhibition by risdiplam occurs in the intestine rather than the liver. The PBPK-predicted risdiplam CYP3A inhibition risk in pediatric patients with SMA aged 2 months-18 years was negligible (midazolam AUCR of 1.09-1.18) and included in the US prescribing information of risdiplam. Comprehensive evaluation of the sensitivity of predicted CYP3A DDI on selected intestinal and hepatic CYP3A ontogeny functions, together with PBPK model-based strategy proposed here, aim to guide and facilitate DDI extrapolations in pediatric populations.

Risdiplam in Type 1 Spinal Muscular Atrophy.

BACKGROUND: Type 1 spinal muscular atrophy is a rare, progressive neuromuscular disease that is caused by low levels of functional survival of motor neuron (SMN) protein. Risdiplam is an orally administered, small molecule that modifies SMN2 pre-messenger RNA splicing and increases levels of functional SMN protein. METHODS: We report the results of part 1 of a two-part, phase 2-3, open-label study of risdiplam in infants 1 to 7 months of age who had type 1 spinal muscular atrophy, which is characterized by the infant not attaining the ability to sit without support. Primary outcomes were safety, pharmacokinetics, pharmacodynamics (including the blood SMN protein concentration), and the selection of the risdiplam dose for part 2 of the study. Exploratory outcomes included the ability to sit without support for at least 5 seconds. RESULTS: A total of 21 infants were enrolled. Four infants were in a low-dose cohort and were treated with a final dose at month 12 of 0.08 mg of risdiplam per kilogram of body weight per day, and 17 were in a high-dose cohort and were treated with a final dose at month 12 of 0.2 mg per kilogram per day. The baseline median SMN protein concentrations in blood were 1.31 ng per milliliter in the low-dose cohort and 2.54 ng per milliliter in the high-dose cohort; at 12 months, the median values increased to 3.05 ng per milliliter and 5.66 ng per milliliter, respectively, which represented a median of 3.0 times and 1.9 times the baseline values in the low-dose and high-dose cohorts, respectively. Serious adverse events included pneumonia, respiratory tract infection, and acute respiratory failure. At the time of this publication, 4 infants had died of respiratory complications. Seven infants in the high-dose cohort and no infants in the low-dose cohort were able to sit without support for at least 5 seconds. The higher dose of risdiplam (0.2 mg per kilogram per day) was selected for part 2 of the study. CONCLUSIONS: In infants with type 1 spinal muscular atrophy, treatment with oral risdiplam led to an increased expression of functional SMN protein in the blood. (Funded by F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT02913482.).

Wear-Off of OnabotulinumtoxinA Effect Over the Treatment Interval in Chronic Migraine: A Retrospective Chart Review With Analysis of Headache Diaries.

OBJECTIVE: To quantify wear-off of the response to OnabotulinumtoxinA (OnabotA) treatment over the treatment cycle in chronic migraine at group and individual level. BACKGROUND: OnabotA administered quarterly is an effective treatment for chronic migraine. However, some patients report that headache recurs before the scheduled follow-up injection. METHODS: In this retrospective chart review performed in 6 university outpatient centers or private practices specialized in headache treatment, 112 patients with a ≥30% response to OnabotA who completed headache diaries over 13 weeks after OnabotA treatment were included (age [mean ± SD] 45 ± 12 years, 82% female, headache days/month at baseline 24 ± 6). RESULTS: Compared to weeks 5 to 8 after injection, headache days/week increased significantly in weeks 12 (+0.52 ± 1.96, 95% CI [0.15, 0.88], P < .009) and 13 (+1.15 ± 1.95, CI[0.79, 1.52], P < .001), demonstrating significant wear-off of the OnabotA effect. Similarly, acute medication days/week significantly increased in weeks 12 (0.38±1.67, CI [0.06, 0.69], P ≤ .027) and 13 (+0.83 ± 1.76, CI [0.49, 1.16], P < .001). At an individual level, 57 patients (51%) showed ≥30% wear-off by weeks 12 and 13, and 28 patients (25%) showed ≥30% wear-off already by weeks 10 and 11. Age, gender, OnabotA dose or cycle number, or headache center did not predict individual wear-off. CONCLUSIONS: These data show that in clinical practice, on average the response of chronic migraine patients to OnabotA injection shows a clinically significant wear-off from week 12 after treatment. About 25% of the patients experience wear-off even by weeks 10 and 11. It must be noted that wear-off detected in a real-world study on OnabotA responders can be due to wear-off of a pharmacological OnabotA effect or a placebo effect, or to regression to the mean effects. This wear-off phenomenon may negatively affect quality of life of chronic migraine patients under OnabotA treatment. The best way to counteract wear-off remains to be determined.

Rimeporide as a first- in-class NHE-1 inhibitor: Results of a phase Ib trial in young patients with Duchenne Muscular Dystrophy.

Rimeporide, a first-in-class sodium/proton exchanger Type 1 inhibitor (NHE-1 inhibitor) is repositioned by EspeRare for patients with Duchenne Muscular Dystrophy (DMD). Historically, NHE-1 inhibitors were developed for cardiac therapeutic interventions. There is considerable overlap in the pathophysiological mechanisms in Congestive Heart Failure (CHF) and in cardiomyopathy in DMD, therefore NHE-1 inhibition could be a promising pharmacological approach to the cardiac dysfunctions observed in DMD. Extensive preclinical data was collected in various animal models including dystrophin-deficient (mdx) mice to characterise Rimeporide's anti-fibrotic and anti-inflammatory properties and there is evidence that NHE-1 inhibitors could play a significant role in modifying DMD cardiac and also skeletal pathologies, as the NHE-1 isoform is ubiquitous. We report here the first study with Rimeporide in DMD patients. This 4-week treatment, open label phase Ib, multiple oral ascending dose study, enrolled 20 ambulant boys with DMD (6-11 years), with outcomes including safety, pharmacokinetic (PK) and pharmacodynamic (PD) biomarkers. Rimeporide was safe and well-tolerated at all doses. PK evaluations showed that Rimeporide was well absorbed orally reaching pharmacological concentrations from the lowest dose, with exposure increasing linearly with dose and with no evidence of accumulation upon repeated dosing. Exploratory PD biomarkers showed positive effect upon a 4-week treatment, supporting its therapeutic potential in patients with DMD, primarily as a cardioprotective treatment, and provide rationale for further efficacy studies.

Leveraging Quantitative Systems Pharmacology Approach into Development of Human Recombinant Follistatin Fusion Protein for Duchenne Muscular Dystrophy.

Quantitative understanding about the dynamics of drug-target interactions in biological systems is essential, especially in rare disease programs with small patient populations. Follistatin, by antagonism of myostatin and activin, which are negative regulators of skeletal muscle and inflammatory response, is a promising therapeutic target for Duchenne Muscular Dystrophy. In this study, we constructed a quantitative systems pharmacology model for FS-EEE-Fc, a follistatin recombinant protein to investigate its efficacy from dual target binding, and, subsequently, to project its human efficacious dose. Based on model simulations, with an assumed efficacy threshold of 7-10% muscle volume increase, 3-5 mg/kg weekly dosing of FS-EEE-Fc is predicted to achieve meaningful clinical outcome. In conclusion, the study demonstrated an application of mechanism driven approach at early stage of a rare disease drug development to support lead compound optimization, enable human dose, pharmacokinetics, and efficacy predictions.

Eficacia de pregabalina, gabapentina y duloxetina en el dolor neuropático verificado por el análisis de la curva-p / Efficacy of pregabalin, gabapentin and duloxetine in neuropathic pain verified by the p-curve analysis

INTRODUCCIÓN: Existe una preocupación creciente entre los clínicos y los investigadores de que muchos resultados publicados en revistas científicas se tratan de falsos positivos. OBJETIVO: Determinar el valor de evidencia o integridad del cuerpo de la literatura publicada sobre la eficacia de pregabalina, gabapentina y duloxetina en el tratamiento del dolor neuropático. MÉTODOS: Se realizó una búsqueda bibliográfica seleccionando ensayos clínicos aleatorizados que evaluaban la eficacia de pregabalina, gabapentina y duloxetina en dolor neuropático. Se aplicó el análisis de curva-p de los estudios con resultados estadísticamente significativos para estudiar su distribución. RESULTADOS: Se demostró que existía una asimetría significativa a la derecha en la curva-p de los tres fármacos (test continuo p < 0,0001) confirmando el valor de evidencia de los estudios. CONCLUSIONES: Los clínicos, los científicos y las publicaciones científicas deben ser conscientes del problema creciente con el "p-hacking" y sus efectos perjudiciales. Todas las partes comparten la responsabilidad en mantener la integridad científica de la literatura publicada

INTRODUCTION: There is a growing concern among clinicians and researchers that many results published in scientific journals are false positives. OBJECTIVE: To determine the value of evidence or integrity of the body of the published literature on the efficacy of pregabalin, gabapentin and duloxetine in the treatment of neuropathic pain. METHODS: A literature search was conducted selecting randomized clinical trials that evaluated the efficacy of pregabalin, gabapentin and duloxetine in neuropathic pain. The p-curve analysis of the studies with statistically significant results was applied to study their distribution. RESULTS: It was demonstrated that there was a significant asymmetry to the right in the p-curve of the three drugs (continuous test p <0.0001) confirming the value of evidence from the studies. CONCLUSIONS: Clinicians, scientists and scientific publications should be aware of the growing problem with "p-hacking" and its harmful effects. All parties share the responsibility to maintain the scientific integrity of the published literature

Pharmacokinetics and tissue distribution of 3,4-diaminopyridine in rats.

Lambert-Eaton myasthenic syndrome (LEMS) is characterized by muscle weakness, amyotrophy, easy fatigability, and depressed tendon reflexes. 3,4-Diaminopyridine (3,4-DAP) is the recommended therapy for the treatment of LEMS. However, estimations of 3,4-DAP pharmacokinetics in human and animals, such as rats, are rarely reported because 3,4-DAP is an orphan drug for the treatment of a very rare disease (LEMS). In particular, little is known about its tissue distribution. Therefore, the pharmacokinetics of 3,4-DAP were studied, with particular focus on tissue distribution, in rats. After intravenous administration of 3,4-DAP to rats, the half-life of 3,4-DAP was 15.9 ± 3.9 min and the volume of distribution at steady-state was 2.8 ± 0.7 L/kg. The tissue-to-plasma partition coefficient (Kp) was high in the kidney, heart, and muscle. In addition, with increased steady state plasma concentration (Css), a tendency toward increased Kp was found in most tissues. In the muscle, a likely target region of 3,4-DAP in LEMS patients, the Kp was higher than in the plasma. Furthermore, more than 68% of 3,4-DAP was distributed to the muscle as determined by the ratio of 3,4-DAP distribution calculated from the apparent volumes of distribution. Hence, 3,4-DAP may provide for more effective and long-lasting effects.

Phase 1 Study of Edasalonexent (CAT-1004), an Oral NF-κB Inhibitor, in Pediatric Patients with Duchenne Muscular Dystrophy.

BACKGROUND: Edasalonexent is an orally administered small molecule designed to inhibit NF-κB, which is activated from infancy in Duchenne muscular dystrophy and is central to causing muscle damage and preventing muscle regeneration. OBJECTIVE: Evaluate the safety, tolerability, pharmacokinetics and exploratory pharmacodynamics of three doses of edasalonexent in ambulatory males ≥4 to <8 years of age with genetically confirmed Duchenne muscular dystrophy. METHODS: This was a 1-week, open-label, multiple-dose study with 3 sequential ascending doses (33, 67 and 100 mg/kg/day) of edasalonexent administered under different dietary conditions to 17 males with a mean age of 5.5 years. RESULTS: All doses of edasalonexent were well tolerated, with no serious adverse events, no drug discontinuations and no dose reductions. The majority of adverse events were mild, and the most common adverse events were gastrointestinal (primarily diarrhea). Edasalonexent was rapidly absorbed with peak levels observed 2-6 hours after dosing and exposures appeared to increase nearly proportionally to dose for the 2 lower and all 3 doses under low-fat and high-fat meal conditions, respectively. Only minor plasma accumulation of edasalonexent was observed with 7 days of dosing. After treatment with edasalonexent for 7 days, levels of NF-κB-regulated genes and serum proteins were decreased. CONCLUSIONS: This first report of edasalonexent oral administration for one week in male pediatric patients with Duchenne muscular dystrophy showed that treatment was well tolerated and inhibited NF-kB pathways.

A phase 1 healthy male volunteer single escalating dose study of the pharmacokinetics and pharmacodynamics of risdiplam (RG7916, RO7034067), a SMN2 splicing modifier.

AIMS: Risdiplam (RG7916, RO7034067) is an orally administered, centrally and peripherally distributed, survival of motor neuron 2 (SMN2) mRNA splicing modifier for the treatment of spinal muscular atrophy (SMA). The objectives of this entry-into-human study were to assess the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics of risdiplam, and the effect of the strong CYP3A inhibitor itraconazole on the PK of risdiplam in healthy male volunteers. METHODS: Part 1 had a randomized, double-blind, adaptive design with 25 subjects receiving single ascending oral doses of risdiplam (ranging from 0.6-18.0 mg, n = 18) or placebo (n = 7). A Bayesian framework was applied to estimate risdiplam's effect on SMN2 mRNA. The effect of multiple doses of itraconazole on the PK of risdiplam was also assessed using a two-period cross-over design (n = 8). RESULTS: Risdiplam in the fasted or fed state was well tolerated. Risdiplam exhibited linear PK over the dose range with a multi-phasic decline with a mean terminal half-life of 40-69 h. Food had no relevant effect, and itraconazole had only a minor effect on plasma PK indicating a low fraction of risdiplam metabolized by CYP3A. The highest tested dose of 18.0 mg risdiplam led to approximately 41% (95% confidence interval 27-55%) of the estimated maximum increase in SMN2 mRNA. CONCLUSIONS: Risdiplam was well tolerated and proof of mechanism was demonstrated by the intended shift in SMN2 splicing towards full-length SMN2 mRNA. Based on these data, Phase 2/3 studies of risdiplam in patients with SMA are now ongoing.

Evaluation of bilosomes as nanocarriers for transdermal delivery of tizanidine hydrochloride: in vitro and ex vivo optimization.

Bilosomes were developed in order to investigate their efficacy as nanocarriers for transdermal delivery of Tizanidine HCl (TZN), a skeletal muscle relaxant with low oral bioavailability. Full factorial experimental design consisting of 27 combinations was generated to study the effects of surfactant type, surfactant-to-cholesterol ratio and the amount of bile salt on the entrapment efficiency (EE), the vesicle size (VS) and in vitro dissolution of the TZN-loaded bilosomes. The permeation through the stratum cornea was optimized with the vertical diffusion assembly using excised rat skin. The permeation parameters of the selected bilosomes were compared to the unformulated drug and it was shown that TZN-B24 exhibited the highest enhancement ratio (ER = 8.8).The optimal formula (TZN-B24) consisting of span 60 in a ratio with cholesterol of 1:1 and 20 mg of bile salt was obtained by employing the desirability function of Design-Expert® software. The mathematical model used for the optimization was validated by comparing the predicted values of the EE (82.3%) and the VS (165.8 nm) with the experimental values of EE = 84.42% and of VS = 161.95 nm. TZN-B24 displayed high zeta potential which contributed to its good stability. It was evident from the results of this study that incorporating TZN in bilosomes improved significantly its permeation through the skin barrier and thus bilosomes can offer a potential nanoplatform using the transdermal route to improve the bioavailability of the drug.

Risdiplam distributes and increases SMN protein in both the central nervous system and peripheral organs.

Spinal muscular atrophy (SMA) is a rare, inherited neuromuscular disease caused by deletion and/or mutation of the Survival of Motor Neuron 1 (SMN1) gene. A second gene, SMN2, produces low levels of functional SMN protein that are insufficient to fully compensate for the lack of SMN1. Risdiplam (RG7916; RO7034067) is an orally administered, small-molecule SMN2 pre-mRNA splicing modifier that distributes into the central nervous system (CNS) and peripheral tissues. To further explore risdiplam distribution, we assessed in vitro characteristics and in vivo drug levels and effect of risdiplam on SMN protein expression in different tissues in animal models. Total drug levels were similar in plasma, muscle, and brain of mice (n = 90), rats (n = 148), and monkeys (n = 24). As expected mechanistically based on its high passive permeability and not being a human multidrug resistance protein 1 substrate, risdiplam CSF levels reflected free compound concentration in plasma in monkeys. Tissue distribution remained unchanged when monkeys received risdiplam once daily for 39 weeks. A parallel dose-dependent increase in SMN protein levels was seen in CNS and peripheral tissues in two SMA mouse models dosed with risdiplam. These in vitro and in vivo preclinical data strongly suggest that functional SMN protein increases seen in patients' blood following risdiplam treatment should reflect similar increases in functional SMN protein in the CNS, muscle, and other peripheral tissues.

GsMTx4-D provides protection to the D2.mdx mouse.

Duchenne muscular dystrophy is a life-limiting muscle disease that has no current effective therapy. Despite mounting evidence that dysregulation of mechanosensitive ion channels is a significant contributor to dystrophy pathogenesis, effective pharmacologic strategies targeting these channels are lacking. GsMTx4, and its enantiomer GsMTx4-D, are peptide inhibitors of mechanosensitive channels with identical activity. In previous studies, acute in vitro application of GsMTx4 to dystrophic murine muscle effectively reduced the excess MSC dependent calcium influx linked to contraction-induced muscle damage. Here we sought to determine if in vivo treatment with GsMTx4-D proffered benefit in the D2.mdx mouse. GsMTx4-D showed a 1-week half-life when administered by subcutaneous injection over four weeks. Informed by these results, D2.mdx mice were then treated by a subcutaneous injection regimen of GsMTx4-D for six weeks followed by determination of muscle mass, muscle susceptibility to eccentric contraction injury and multiple histological indicators of disease progression. The mice showed a reduction in the loss of muscle mass and a decrease in susceptibility to contraction induced injury. These protective effects were realized without reduction in fibrosis, supporting a model where GsMTx4-D acts directly on muscle cells. We propose GsMTx4-D represents a promising new therapy to slow disease progression and may complement other therapies such as anti-inflammatory agents and gene-replacement strategies.

Development of a Clinically Relevant Dissolution Method for Metaxalone Immediate Release Formulations Based on an IVIVC Model.

PURPOSE: The aim of the present work was to classify metaxalone according to the Biopharmaceutics Classification System (BCS), to develop a clinically relevant dissolution method that can be used to predict the oral absorption of metaxalone and to establish an in vitro-in vivo correlation (IVIVC). METHODS: Solubility of the drug was studied in different pH media and permeability studies were performed using a Caco-2 cell model. The in vitro dissolution and in vivo disposition of metaxalone from 3 different immediate release (IR) tablet formulations were investigated using USP 2 apparatus and a single dose, four-way, crossover bioequivalence study in healthy humans along with an oral solution of the drug, respectively. An IVIVC was established by using a direct, differential based method. RESULTS: Metaxalone has been confirmed as a Class II drug according to BCS. Bioavailability studies performed in humans demonstrated that dissolution was the rate limiting step for bioavailability of the drug and one of the test products had significantly improved bioavailability compared to the marketed product Skelaxin®. An IVIVC model was developed that demonstrated an acceptable internal predictability. CONCLUSION: The IVIVC demonstrated that formulation factors play a significant role in dissolution and absorption of metaxalone. A pH 4.5 dissolution medium containing 0.5% NaCl with 0.2% SLS (USP apparatus 2 at 50 rpm) is clinically relevant to predict bioavailability of the drug and is superior to the USP method in terms of the Quality by Design (QbD) concept.

Role of central versus peripheral opioid system in antinociceptive and anti-inflammatory effect of botulinum toxin type A in trigeminal region.

BACKGROUND: Although botulinum toxin type A (BT-A) is approved for chronic migraine treatment, its site and mechanism of action are still elusive. Recently our group discovered that suppression of CGRP release from dural nerve endings might account for antimigraine action of pericranially injected BT-A. We demonstrated that central antinociceptive effect of BT-A in sciatic region involves endogenous opioid system as well. Here we investigated possible interaction of BT-A with endogenous opioid system within the trigeminal region. METHODS: In orofacial formalin test we investigated the influence of centrally acting opioid antagonist naltrexone (2 mg/kg, s.c.) versus peripherally acting methylnaltrexone (2 mg/kg, s.c.) on BT-A's (5 U/kg, s.c. into whisker pad) or morphine's (6 mg/kg, s.c.) antinociceptive effect and the effect on dural neurogenic inflammation (DNI). DNI was assessed by Evans blue-plasma protein extravasation. RESULTS: Naltrexone abolished the effect of BT-A on pain and dural plasma protein extravasation, whereas peripherally acting methylnaltrexone did not change either BT-A's effect on pain or its effect on dural extravasation. Naltrexone abolished the antinociceptive and anti-inflammatory effects of morphine, as well. However, methylnaltrexone decreased the antinociceptive effect of morphine only partially in the second phase of the test and had no significant effect on morphine-mediated reduction in DNI. CONCLUSIONS: Morphine acts on pain in trigeminal region both peripherally and centrally, whereas the effect on dural plasma protein extravasation seems to be only centrally mediated. However, the interaction of BT-A with endogenous opioid system, with consequent inhibition of nociceptive transmission as well as the DNI, occurs primarily centrally. SIGNIFICANCE: Botulinum toxin type A (BT-A)'s axonal transport and potential transcytosis suggest that its antinociceptive effect might involve diverse neurotransmitters at different sites of trigeminal system. Here we discovered that the reduction in pain and accompanying DNI involves the interaction of BT-A with central endogenous opioid system (probably at the level of trigeminal nucleus caudalis).

Cutaneous iontophoresis of µ-conotoxin CnIIIC-A potent NaV1.4 antagonist with analgesic, anaesthetic and myorelaxant properties.

Cutaneous iontophoretic delivery of µ-conotoxin CnIIIC (XEP), a potent peptide antagonist of the NaV1.4 sodium channel, was investigated using porcine ear skin and validated using human abdominal skin. Initial results demonstrated that cutaneous deposition of XEP following iontophoresis was superior to passive delivery and increased with current density. XEP deposition after iontophoresis at 0.1, 0.3 and 0.5mA/cm2 for 2h and 4h was 22.4±0.4, 34.5±1.4, 57.4±7.6µg/cm2 and 30.6±5.4, 53.9±17.2, 90.9±30.8µg/cm2, respectively (cf. corresponding passive controls - 9.8±1.1 and 16.9±1.0µg/cm2). Moreover, tape-stripping studies showed that XEP was mainly adsorbed on the skin surface when administered passively. Co-iontophoresis of acetaminophen demonstrated that XEP was present in the skin as it significantly reduced convective solvent flow as evidenced by the ∼7-fold decrease in acetaminophen permeation. Shorter duration iontophoresis (15, 30 and 60min) was performed and the effect of current density (0.1, 0.3 and 0.5mA/cm2) and concentration (0.1 and 1mM) investigated. Skin deposition of XEP was already quantifiable after iontophoresis for 15min at the lower concentration. There was no statistically significant difference between XEP deposition in porcine and human skin. Confocal laser scanning microscopy enabled post-iontophoretic visualization of FITC-labelled XEP in the epidermis.

Pre-Clinical Study of a Novel Recombinant Botulinum Neurotoxin Derivative Engineered for Improved Safety.

Cyto-012 is a recombinant derivative of Botulinum neurotoxin Type A (BoNT/A). It primarily differs from wild type (wt) BoNT/A1 in that it incorporates two amino acid substitutions in the catalytic domain of the light chain (LC) metalloprotease (E224 > A and Y366 > A), designed to provide a safer clinical profile. Cyto-012 is specifically internalized into rat cortical and hippocampal neurons, and cleaves Synaptosomal-Associated Protein 25 (SNAP-25), the substrate of wt BoNT/A, but exhibits slower cleavage kinetics and therefore requires a higher absolute dose to exhibit pharmacologic activity. The pharmacodynamics of Cyto-012 and wt BoNT/A have similar onset and duration of action using the Digital Abduction Assay (DAS). Intramuscular LD50 values for Cyto-012 and wt BoNT/A respectively, were 0.63 ug (95% CI = 0.61, 0.66) and 6.22 pg (95% CI = 5.42, 7.02). ED50 values for Cyto-012 and wt BoNT/A were respectively, 0.030 ug (95% CI = 0.026, 0.034) and 0.592 pg (95% CI = 0.488, 0.696). The safety margin (intramuscular LD50/ED50 ratio) for Cyto-012 was found to be improved 2-fold relative to wt BoNT/A (p < 0.001). The DAS response to Cyto-012 was diminished when a second injection was administered 32 days after the first. These data suggest that the safety margin of BoNT/A can be improved by modulating their activity towards SNAP-25.