- Email: [email protected]
incobotulinumtoxina (Xeomin) dose ratio
Abstracts / Toxicon 123 (2016) S2eS90
Conclusions: Treatment of upper and lower limb spasticity with increasing incobotulinumtoxinA doses (up to 800 U) improved QoL over the study duration (3 treatments). Data encourage further evaluation of the impact of incobotulinumtoxinA treatment on QoL in patients with spasticity. Funding: Merz Pharmaceuticals Keywords: Botulinum toxin type A; EQ-5D; IncobotulinumtoxinA; Quality of life; Spasticity 72. HPLC ANALYSIS OF RECOMBINANT BOTULINUM NEUROTOXINS: EXPANDING THE ANALYTIC TOOLBOX Malgorzata Field*, James Cummins, Imran Mir, Andrew Splevins, Andy Hooker. Ipsen Bioinnovation, Abingdon, Oxfordshire, UK * Corresponding author: 102 Park Drive, Milton Park, Abingdon, OX14 4RY, UK. E-mail address:malgorzata.fi[email protected].
Introduction and objectives: High pressure liquid chromatography (HPLC) is a powerful tool widely used in the pharmaceutical industry for measuring purity and detecting impurities in different molecules. Currently a limited number of publications regarding the chromatographic separation of active toxins is available, mostly due to the technical challenge of handling botulinum toxins (BoNTs) and safety concerns. Having found solutions to overcome the safety challenges, here we present HPLC methods developed to monitor different purity attributes such as size and aggregation for various serotypes of recombinant BoNTs and novel retargeted BoNTs (targeted secretion inhibitors [TSI]). Methods: Size exclusion and reversed-phase HPLC analytical techniques have been employed to monitor product size, truncation, shape, and hydrophobicity. Based on the recombinant (r)BoNT/TSI properties, the rBoNT/TSI interacts with the resin of the column during the analysis and is eluted by an appropriate mobile phase. BoNTs are extremely potent and therefore require special containment procedures to be in place for handling and decontamination. This poses unique challenges to equipment setup and consideration of assay design. Prior to introducing any of the new chromatographic separation methods, it was necessary to perform rigorous safety checks and process review. Results: The equipment setup and procedures implemented were shown to allow safe analysis of BoNTs by HPLC. Each chromatographic separation method had to be optimized for each BoNT construct assessed, including column type, gradient, mobile phase composition, reducing agent, and column temperature. The resulting methods were shown to be accurate, precise, and able to detect the relevant impurities, many of which are not detected by more standard methods such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and potency assays. Conclusions: A suite of analytic chromatography methods has been developed allowing assessment of purity and impurity profiles of rBoNTs and TSIs Keywords: Botulinum toxin (BoNT); High pressure liquid chromatography (HPLC); Reversed-phase HPLC (RP-HPLC); Size exclusion HPLC (SE-HPLC) 73. BOTULINUM TOXIN TREATMENT ABOBOTULINUMTOXINA (DYSPORT)/INCOBOTULINUMTOXINA (XEOMIN) DOSE RATIO Nicholas A. Fletcher*, Nicola Crummie. Walton Centre NHS Foundation Trust, Liverpool, UK * Corresponding author: Walton Centre NHS Foundation Trust, Lower Lane, Fazakerley, Liverpool, L9 7LJ, UK. E-mail address:nick.fl[email protected].
Introduction: In 2013 the Walton Centre NHS Foundation Trust changed the dose ratio of abobotulinumtoxinA to incobotulinumtoxinA in patients switching between botulinum neurotoxin (BoNT) formulations. In this study the efficacy of a 4:1 dose was assessed in patients switching from abobotulinumtoxinA to incobotulinumtoxinA. Methods: The overall average dose and dose ratio of abobotulinumtoxinA to incobotulinumtoxinA were calculated for patients with the main
S29
conditions treated in the Trust: blepharospasm, hemifacial spasm, spasticity, cervical or other dystonia; in each year of the study: April 2012March 2013 (before change), April 2013-March 2014 (year of change) and April 2014-March 2015 (post-change). Individual patient dose ratios were calculated for a sample of patients with cervical dystonia. Results: For each year studied, the average dose ratio was 3:1 for blepharospasm and 4:1 for cervical dystonia, hemifacial spasm, and spasticity. For other dystonias the average dose ratios were 4:1 in the first two years and 3:1 post-change. The majority of Walton Center patients receiving BoNT had cervical dystonia. Most of these patients (133/145, 92%) switched BoNT using the recommended 4:1 ratio, which remained effective in 111/133 (84%). The 4;1 dose was ineffective in one patient and was gradually increased to 2:1. One patient receiving a 5:1 dose reported “major benefit” with both formulations. A “major” or “moderate benefit” was reported by 45% and 43% of patients receiving abobotulinumtoxinA or incobotulinumtoxinA, respectively. Internal pharmacy calculations (2013) suggested that savings are made if >70% of patients switch at either 4:1 or 3:1. Conclusions: A switch from abobotulinumtoxinA to incobotulinumtoxinA at a 4:1 dose ratio is effective in most cases. In some, an increase to 3:1 is required. The switch to incobotulinumtoxinA was long lasting, the 4:1 ratio was maintained in most, and the switch appeared to be cost effective. Editorial support funded by Merz Pharmaceuticals. Keywords: AbobotulinumtoxinA; Botulinum toxin; Cervical dystonia; IncobotulinumtoxinA 74. USE OF HIGH-CONTENT IMAGING IN BOTULINUM TOXIN RESEARCH Elena Fonfria a, *, Sarah Donald a, Verity A. Cadd a, Johannes Krupp b. Ipsen Bioinnovation, Abingdon, UK; b Ipsen Innovation, Les Ulis, France
a
* Corresponding author: Ipsen Bioinnovation, 102 Park Drive, Milton Park, Abingdon, Oxon, OX14 4RY, UK. E-mail address:[email protected].
Introduction and objectives: In vitro characterization of botulinum neurotoxin (BoNT) activity has traditionally been based on biochemical determination of the cleaved product, which is the last step in its mechanism of action (MOA). The objectives of this work were to: (1) establish a readout in which the localization of BoNTs and BoNT derivatives, with the binding domain replaced with an alternative binding domain (targeted secretion inhibitors; TSI)), can be tracked during cellular intoxication; and (2) compare the results with those from biochemical methods. Methods: Cortical neurons (CTX) were prepared from CD rats using approved procedures. SiMa cells were sourced from DSMZ, Germany. Cells were treated with native (List Biological Laboratories, USA) or recombinant (Ipsen Bioinnovation) BoNT/A, or TSI. Cleavage of synaptosomal-associated protein 25 kDa (SNAP)-25 was determined by Western blot. Localization of the BoNT/A light chain was assessed by high-content imaging (HCI) using the ImageXpress Micro XLS (Molecular Devices, UK). Results: CTX neurons were highly sensitive to BoNT activity. BoNT/A entered CTX in a concentration- and time-dependent manner, and activity was detected in the cytosol within 1 hour. HCI confirmed that BoNT/A entered the cells in compartments that gave a punctate staining pattern, consistent with vesicular endocytosis. SiMa cells were sensitive to both BoNT/A and a BoNT/A-based TSI containing an epidermal growth factor receptor (EGFR)-targeting moiety. Statistically significant BoNT activity in the cytosol was detected for both proteins at 24 hours. BoNT/A internalized in a compartment consistent with synaptic vesicles, whereas the TSI entered a compartment consistent with early endosomes. For both cell systems, internalized BoNT/A could be quantified at earlier time points than biochemical quantification of the cleaved product, thus suggesting effective monitoring of an earlier MOA step. Conclusions: HCI allows automated quantification of BoNT and TSI in a range of cell types. Different endocytic routes leading to the same cleavage product endpoint can be studied in a high-throughput and user-friendly manner. Keywords: BoNT/A1; Botulinum neurotoxin; High-content imaging; In vitro cellular assay; Targeted secretion inhibitors; TSI
Conclusions: Treatment of upper and lower limb spasticity with increasing incobotulinumtoxinA doses (up to 800 U) improved QoL over the study duration (3 treatments). Data encourage further evaluation of the impact of incobotulinumtoxinA treatment on QoL in patients with spasticity. Funding: Merz Pharmaceuticals Keywords: Botulinum toxin type A; EQ-5D; IncobotulinumtoxinA; Quality of life; Spasticity 72. HPLC ANALYSIS OF RECOMBINANT BOTULINUM NEUROTOXINS: EXPANDING THE ANALYTIC TOOLBOX Malgorzata Field*, James Cummins, Imran Mir, Andrew Splevins, Andy Hooker. Ipsen Bioinnovation, Abingdon, Oxfordshire, UK * Corresponding author: 102 Park Drive, Milton Park, Abingdon, OX14 4RY, UK. E-mail address:malgorzata.fi[email protected].
Introduction and objectives: High pressure liquid chromatography (HPLC) is a powerful tool widely used in the pharmaceutical industry for measuring purity and detecting impurities in different molecules. Currently a limited number of publications regarding the chromatographic separation of active toxins is available, mostly due to the technical challenge of handling botulinum toxins (BoNTs) and safety concerns. Having found solutions to overcome the safety challenges, here we present HPLC methods developed to monitor different purity attributes such as size and aggregation for various serotypes of recombinant BoNTs and novel retargeted BoNTs (targeted secretion inhibitors [TSI]). Methods: Size exclusion and reversed-phase HPLC analytical techniques have been employed to monitor product size, truncation, shape, and hydrophobicity. Based on the recombinant (r)BoNT/TSI properties, the rBoNT/TSI interacts with the resin of the column during the analysis and is eluted by an appropriate mobile phase. BoNTs are extremely potent and therefore require special containment procedures to be in place for handling and decontamination. This poses unique challenges to equipment setup and consideration of assay design. Prior to introducing any of the new chromatographic separation methods, it was necessary to perform rigorous safety checks and process review. Results: The equipment setup and procedures implemented were shown to allow safe analysis of BoNTs by HPLC. Each chromatographic separation method had to be optimized for each BoNT construct assessed, including column type, gradient, mobile phase composition, reducing agent, and column temperature. The resulting methods were shown to be accurate, precise, and able to detect the relevant impurities, many of which are not detected by more standard methods such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and potency assays. Conclusions: A suite of analytic chromatography methods has been developed allowing assessment of purity and impurity profiles of rBoNTs and TSIs Keywords: Botulinum toxin (BoNT); High pressure liquid chromatography (HPLC); Reversed-phase HPLC (RP-HPLC); Size exclusion HPLC (SE-HPLC) 73. BOTULINUM TOXIN TREATMENT ABOBOTULINUMTOXINA (DYSPORT)/INCOBOTULINUMTOXINA (XEOMIN) DOSE RATIO Nicholas A. Fletcher*, Nicola Crummie. Walton Centre NHS Foundation Trust, Liverpool, UK * Corresponding author: Walton Centre NHS Foundation Trust, Lower Lane, Fazakerley, Liverpool, L9 7LJ, UK. E-mail address:nick.fl[email protected].
Introduction: In 2013 the Walton Centre NHS Foundation Trust changed the dose ratio of abobotulinumtoxinA to incobotulinumtoxinA in patients switching between botulinum neurotoxin (BoNT) formulations. In this study the efficacy of a 4:1 dose was assessed in patients switching from abobotulinumtoxinA to incobotulinumtoxinA. Methods: The overall average dose and dose ratio of abobotulinumtoxinA to incobotulinumtoxinA were calculated for patients with the main
S29
conditions treated in the Trust: blepharospasm, hemifacial spasm, spasticity, cervical or other dystonia; in each year of the study: April 2012March 2013 (before change), April 2013-March 2014 (year of change) and April 2014-March 2015 (post-change). Individual patient dose ratios were calculated for a sample of patients with cervical dystonia. Results: For each year studied, the average dose ratio was 3:1 for blepharospasm and 4:1 for cervical dystonia, hemifacial spasm, and spasticity. For other dystonias the average dose ratios were 4:1 in the first two years and 3:1 post-change. The majority of Walton Center patients receiving BoNT had cervical dystonia. Most of these patients (133/145, 92%) switched BoNT using the recommended 4:1 ratio, which remained effective in 111/133 (84%). The 4;1 dose was ineffective in one patient and was gradually increased to 2:1. One patient receiving a 5:1 dose reported “major benefit” with both formulations. A “major” or “moderate benefit” was reported by 45% and 43% of patients receiving abobotulinumtoxinA or incobotulinumtoxinA, respectively. Internal pharmacy calculations (2013) suggested that savings are made if >70% of patients switch at either 4:1 or 3:1. Conclusions: A switch from abobotulinumtoxinA to incobotulinumtoxinA at a 4:1 dose ratio is effective in most cases. In some, an increase to 3:1 is required. The switch to incobotulinumtoxinA was long lasting, the 4:1 ratio was maintained in most, and the switch appeared to be cost effective. Editorial support funded by Merz Pharmaceuticals. Keywords: AbobotulinumtoxinA; Botulinum toxin; Cervical dystonia; IncobotulinumtoxinA 74. USE OF HIGH-CONTENT IMAGING IN BOTULINUM TOXIN RESEARCH Elena Fonfria a, *, Sarah Donald a, Verity A. Cadd a, Johannes Krupp b. Ipsen Bioinnovation, Abingdon, UK; b Ipsen Innovation, Les Ulis, France
a
* Corresponding author: Ipsen Bioinnovation, 102 Park Drive, Milton Park, Abingdon, Oxon, OX14 4RY, UK. E-mail address:[email protected].
Introduction and objectives: In vitro characterization of botulinum neurotoxin (BoNT) activity has traditionally been based on biochemical determination of the cleaved product, which is the last step in its mechanism of action (MOA). The objectives of this work were to: (1) establish a readout in which the localization of BoNTs and BoNT derivatives, with the binding domain replaced with an alternative binding domain (targeted secretion inhibitors; TSI)), can be tracked during cellular intoxication; and (2) compare the results with those from biochemical methods. Methods: Cortical neurons (CTX) were prepared from CD rats using approved procedures. SiMa cells were sourced from DSMZ, Germany. Cells were treated with native (List Biological Laboratories, USA) or recombinant (Ipsen Bioinnovation) BoNT/A, or TSI. Cleavage of synaptosomal-associated protein 25 kDa (SNAP)-25 was determined by Western blot. Localization of the BoNT/A light chain was assessed by high-content imaging (HCI) using the ImageXpress Micro XLS (Molecular Devices, UK). Results: CTX neurons were highly sensitive to BoNT activity. BoNT/A entered CTX in a concentration- and time-dependent manner, and activity was detected in the cytosol within 1 hour. HCI confirmed that BoNT/A entered the cells in compartments that gave a punctate staining pattern, consistent with vesicular endocytosis. SiMa cells were sensitive to both BoNT/A and a BoNT/A-based TSI containing an epidermal growth factor receptor (EGFR)-targeting moiety. Statistically significant BoNT activity in the cytosol was detected for both proteins at 24 hours. BoNT/A internalized in a compartment consistent with synaptic vesicles, whereas the TSI entered a compartment consistent with early endosomes. For both cell systems, internalized BoNT/A could be quantified at earlier time points than biochemical quantification of the cleaved product, thus suggesting effective monitoring of an earlier MOA step. Conclusions: HCI allows automated quantification of BoNT and TSI in a range of cell types. Different endocytic routes leading to the same cleavage product endpoint can be studied in a high-throughput and user-friendly manner. Keywords: BoNT/A1; Botulinum neurotoxin; High-content imaging; In vitro cellular assay; Targeted secretion inhibitors; TSI