- Email: [email protected]
HPLC analysis of recombinant botulinum neurotoxins: Expanding the analytic toolbox
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Abstracts / Toxicon 123 (2016) S2eS90
Conclusion: Impairment and symptoms-related goals predominated as primary goals in our population. The majority of patients achieved the goals established for the treatment cycle. GAS proved to be a useful and sensitive tool to measure treatment outcome with the focus on the needs and expectations of patients/carers, establishing realistic and clear treatment goals, and leading to good achievement rates. Keywords: Botulinum toxin; Hemorrhagic stroke; Spasticity; Goal Attainment Scaling 69. NEUROGENIC TEMPOROMANDIBULAR JOINT DISLOCATIONS TREATED WITH BOTULINUM TOXIN INJECTIONS: CASE REPORT ~o*, Isabel Amorim, Sofia Proença, Jorge Jacinto. Carolina Falca ~o de Alcoita ~o, Serviço de Reabilitaça ~o de Centro de Medicina de Reabilitaça Adultos 3, Alcabideche, Portugal ~o de Alcoit~ * Corresponding author: Centro de Medicina de Reabilitaça ao, Rua ~o. 2649-506 Alcabideche, Portugal. E-mail address:caroConde Bar~ ao e Alcoita [email protected].
Introduction: Temporomandibular joint (TMJ) dislocations that occur as a result of an increased tone in the protractor masticatory muscles were recently defined as neurogenic dislocations of the TMJ. Recurrent dislocation of the mandibular condyle presents a difficult problem for patients. Dislocations become more frequent and more difficult to avoid. While conservative treatment is often not sufficient and surgical procedures are invasive, botulinum toxin type A (BTX-A) injections into the lateral pterygoid muscles, offer an option for a period of time without renewed dislocation. Methods: Case report and literature review about the evidence for BTX-A injections into the lateral pterygoid muscles in recurrent neurogenic dislocations of the TMJ. Research was conducted in PubMed and Medline databases, and 5 of 26 papers were selected containing relevant information. Results: A 36-year-old woman was admitted at a rehabilitation center, with posthemorrhagic stroke and quadriparesis (no functionality at all), cognitive deficit, dysphagia, dysarthria, and incontinence. She also presented with recurrent episodes of oromandibular dystonia causing painful and disabling TMJ dislocation, interfering with the rehabilitation program and quality of life. Intermaxillary fixation screws were placed for mandibular block and conservative measures were implemented. After maxillofacial surgeons excluded surgery as an option, 3 months later, TMJ dislocation episodes continued (2-3 daily). So BTX-A injection was applied to both lateral pterygoid muscles, and these episodes ceased to happen from 24 h after the injection till the date of this abstract (62 days). Discussion/Conclusion: Due to BTXA treatment, our patient became more capable of participating in the rehabilitation program. BTX-A injection was effective in neurogenic TMJ dislocation, with no side effects, improving the patient’s speech, head posture, mastication, and mood and motivation for at least 62 days. Keywords: Botulinum toxin; Dislocation; Hemorrhagic stroke; TMJ 70. IMPROVEMENT OF TINNITUS AFTER TREATMENT OF HEMIFACIAL SPASM WITH BOTULINUM TOXIN ao b, Diogo Raposo c, Margarida Mariana Valente Fernandes a,*, Leonor Rebord~ Vargas c, Patrícia Pita Lobo b, Cristina Costa b. a Neurology Department, Instituto Portugu^ es de Oncologia, Lisboa, Portugal; b Neurology Department, Hospital Fernando Fonseca, Amadora/Sintra, Portugal; c Otolaryngology Department, Hospital Fernando Fonseca, Amadora/ Sintra, Portugal ^s de Oncologia, * Corresponding author: Neurology Department, Instituto Portugue Rua Professor Lima Basto, 1099-023 Lisboa, Portugal. E-mail address:[email protected].
Introduction and objectives: Hemifacial spasm (HFS) is characterized by unilateral involuntary contraction of muscles supplied by the facial nerve. It is frequently accompanied by nonmotor symptoms, such as tinnitus, which are often overlooked. The underlying pathophysiology is both complex and controversial. The aim of this open-label study was to identify and characterize patients with HFS and associated tinnitus.
Methods: The occurrence and features of tinnitus associated with HFS were assessed by a questionnaire to patients who were followed and treated in a botulinum toxin (BT) outpatient clinic, between August 2015 and August 2016. Patients were apprised regarding the relevance of tinnitus and its degree of disability compared with that of facial spasm. They were evaluated before and after a BT treatment, using the Hemifacial Spasm-30, Tinnitus Functional Index and Short Form Health Survey 36 Item v2 questionnaires. Otologic examination and auditory evoked potentials test were performed. The aim of BT treatment was relief of HFS in all cases. Results: Of 35 patients followed, 31 were included and four patients with tinnitus associated with HFS were identified: 1 male, 3 females, ages between 45 and 61 years. All had tinnitus simultaneous and ipsilateral to the spasm. In 2 patients, HFS was secondary to neurovascular compression, and the other 2 had a previous history of Bell’s palsy. One patient had bilateral HFS and tinnitus. Three patients stated that the tinnitus was as disturbing as the HFS, and 1 stated that it was more incapacitating than HFS. Three patients were treated with Dysport (average dose/HFS 112.5 U), and 1 patient with Botox (dose 67.5 U). There was improvement of both HFS and tinnitus with treatment in all patients. Conclusions: Tinnitus may be an important source of disability in HFS patients, and its presence should therefore be screened for. Treatment with BT may be effective not only in HFS but also in coexisting tinnitus. Keywords: Botulinum toxin treatment; Hemifacial spasm; Tinnitus 71. INCOBOTULINUMTOXINA (XEOMIN) TREATMENT IMPROVES QUALITY OF LIFE OF PATIENTS WITH UPPER AND LOWER LIMB SPASTICITY Klemens Fheodoroff a,*, Tiina Rekand b, Luisa Medeiros c, Peter € rg Wissel e, Djamel Bensmail f, Astrid Scheschonka g, Birgit Kossmehl d, Jo Flatau-Baque g, Olivier Simon g, Dirk Dressler h,1, David Simpson i,1. a Gailtal-Klinik, Hermagor, Austria; b Haukeland University Hospital, Bergen, Norway; c Centro Hospitalar de Lisboa Central, Lisboa, Portugal; d Vivantes Humboldt-Klinikum, Berlin, Germany; e Vivantes Hospital Spandau, Berlin, e Hospital, AP-HP, University of Versailles Saint Germany; f Raymond-Poincar Quentin, Garches, France; g Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany; h Hannover Medical School, Hannover, Germany; i Icahn School of Medicine at Mount Sinai, New York, NY, USA * Corresponding author: Gailtal-Klinik, Radniger Strasse 12, A-9620 Hermagor, Austria. E-mail address:[email protected].
Introduction and objectives: The safety and efficacy of botulinum toxin treatment for limb spasticity are well established. However, reports on the effect of treatment on quality of life (QoL) are inconclusive. Here we report QoL outcomes following incobotulinumtoxinA (Xeomin) treatment of upper and lower limb spasticity in the TOWER study. Methods: The open-label TOWER study (NCT01603459) assessed the safety and efficacy of escalating incobotulinumtoxinA doses (400 U, 600 U, and 600-800 U) in patients (ages 18-80 years) with upper and lower limb spasticity on the same body side due to stroke or other cerebral causes. QoL was assessed using the EQ-5D questionnaire, which includes 5 dimensions (mobility, self-care, usual activities, pain/discomfort, anxiety/depression), and a visual analog scale (VAS) on which patients rate their current state of health from 0 (worst) to 100 (best). Results: Patients (N¼155) were enrolled and received incobotulinumtoxinA. For all EQ-5D dimensions, the proportion of patients with improvements from study baseline to end of injection cycle (IC) 3 was higher than the proportion with worsening: mobility, 11.4% vs 2.9%; self-care, 21.4% vs 7.1%; usual activities, 24.3% vs 5.0%; pain/discomfort, 30.7% vs 9.3%; anxiety/depression, 25.0% vs 9.3%. The mean (standard deviation; SD) change in EQ-5D VAS score from study baseline to 4 weeks post-treatment was 6.7 (14.1) points, 9.6 (16.3) points, and 8.6 (17.0) points in IC1, IC2, and IC3, respectively (P<0.0001 for all). The mean (SD) EQ-5D VAS score improved from 59.9 (18.9) at baseline to 70.5 (16.7) at the end of IC3, corresponding to a mean (SD) change of 10.5 (17.5) points (P<0.0001).
1
DD and DS contributed equally to this abstract.
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
Abstracts / Toxicon 123 (2016) S2eS90
Conclusion: Impairment and symptoms-related goals predominated as primary goals in our population. The majority of patients achieved the goals established for the treatment cycle. GAS proved to be a useful and sensitive tool to measure treatment outcome with the focus on the needs and expectations of patients/carers, establishing realistic and clear treatment goals, and leading to good achievement rates. Keywords: Botulinum toxin; Hemorrhagic stroke; Spasticity; Goal Attainment Scaling 69. NEUROGENIC TEMPOROMANDIBULAR JOINT DISLOCATIONS TREATED WITH BOTULINUM TOXIN INJECTIONS: CASE REPORT ~o*, Isabel Amorim, Sofia Proença, Jorge Jacinto. Carolina Falca ~o de Alcoita ~o, Serviço de Reabilitaça ~o de Centro de Medicina de Reabilitaça Adultos 3, Alcabideche, Portugal ~o de Alcoit~ * Corresponding author: Centro de Medicina de Reabilitaça ao, Rua ~o. 2649-506 Alcabideche, Portugal. E-mail address:caroConde Bar~ ao e Alcoita [email protected].
Introduction: Temporomandibular joint (TMJ) dislocations that occur as a result of an increased tone in the protractor masticatory muscles were recently defined as neurogenic dislocations of the TMJ. Recurrent dislocation of the mandibular condyle presents a difficult problem for patients. Dislocations become more frequent and more difficult to avoid. While conservative treatment is often not sufficient and surgical procedures are invasive, botulinum toxin type A (BTX-A) injections into the lateral pterygoid muscles, offer an option for a period of time without renewed dislocation. Methods: Case report and literature review about the evidence for BTX-A injections into the lateral pterygoid muscles in recurrent neurogenic dislocations of the TMJ. Research was conducted in PubMed and Medline databases, and 5 of 26 papers were selected containing relevant information. Results: A 36-year-old woman was admitted at a rehabilitation center, with posthemorrhagic stroke and quadriparesis (no functionality at all), cognitive deficit, dysphagia, dysarthria, and incontinence. She also presented with recurrent episodes of oromandibular dystonia causing painful and disabling TMJ dislocation, interfering with the rehabilitation program and quality of life. Intermaxillary fixation screws were placed for mandibular block and conservative measures were implemented. After maxillofacial surgeons excluded surgery as an option, 3 months later, TMJ dislocation episodes continued (2-3 daily). So BTX-A injection was applied to both lateral pterygoid muscles, and these episodes ceased to happen from 24 h after the injection till the date of this abstract (62 days). Discussion/Conclusion: Due to BTXA treatment, our patient became more capable of participating in the rehabilitation program. BTX-A injection was effective in neurogenic TMJ dislocation, with no side effects, improving the patient’s speech, head posture, mastication, and mood and motivation for at least 62 days. Keywords: Botulinum toxin; Dislocation; Hemorrhagic stroke; TMJ 70. IMPROVEMENT OF TINNITUS AFTER TREATMENT OF HEMIFACIAL SPASM WITH BOTULINUM TOXIN ao b, Diogo Raposo c, Margarida Mariana Valente Fernandes a,*, Leonor Rebord~ Vargas c, Patrícia Pita Lobo b, Cristina Costa b. a Neurology Department, Instituto Portugu^ es de Oncologia, Lisboa, Portugal; b Neurology Department, Hospital Fernando Fonseca, Amadora/Sintra, Portugal; c Otolaryngology Department, Hospital Fernando Fonseca, Amadora/ Sintra, Portugal ^s de Oncologia, * Corresponding author: Neurology Department, Instituto Portugue Rua Professor Lima Basto, 1099-023 Lisboa, Portugal. E-mail address:[email protected].
Introduction and objectives: Hemifacial spasm (HFS) is characterized by unilateral involuntary contraction of muscles supplied by the facial nerve. It is frequently accompanied by nonmotor symptoms, such as tinnitus, which are often overlooked. The underlying pathophysiology is both complex and controversial. The aim of this open-label study was to identify and characterize patients with HFS and associated tinnitus.
Methods: The occurrence and features of tinnitus associated with HFS were assessed by a questionnaire to patients who were followed and treated in a botulinum toxin (BT) outpatient clinic, between August 2015 and August 2016. Patients were apprised regarding the relevance of tinnitus and its degree of disability compared with that of facial spasm. They were evaluated before and after a BT treatment, using the Hemifacial Spasm-30, Tinnitus Functional Index and Short Form Health Survey 36 Item v2 questionnaires. Otologic examination and auditory evoked potentials test were performed. The aim of BT treatment was relief of HFS in all cases. Results: Of 35 patients followed, 31 were included and four patients with tinnitus associated with HFS were identified: 1 male, 3 females, ages between 45 and 61 years. All had tinnitus simultaneous and ipsilateral to the spasm. In 2 patients, HFS was secondary to neurovascular compression, and the other 2 had a previous history of Bell’s palsy. One patient had bilateral HFS and tinnitus. Three patients stated that the tinnitus was as disturbing as the HFS, and 1 stated that it was more incapacitating than HFS. Three patients were treated with Dysport (average dose/HFS 112.5 U), and 1 patient with Botox (dose 67.5 U). There was improvement of both HFS and tinnitus with treatment in all patients. Conclusions: Tinnitus may be an important source of disability in HFS patients, and its presence should therefore be screened for. Treatment with BT may be effective not only in HFS but also in coexisting tinnitus. Keywords: Botulinum toxin treatment; Hemifacial spasm; Tinnitus 71. INCOBOTULINUMTOXINA (XEOMIN) TREATMENT IMPROVES QUALITY OF LIFE OF PATIENTS WITH UPPER AND LOWER LIMB SPASTICITY Klemens Fheodoroff a,*, Tiina Rekand b, Luisa Medeiros c, Peter € rg Wissel e, Djamel Bensmail f, Astrid Scheschonka g, Birgit Kossmehl d, Jo Flatau-Baque g, Olivier Simon g, Dirk Dressler h,1, David Simpson i,1. a Gailtal-Klinik, Hermagor, Austria; b Haukeland University Hospital, Bergen, Norway; c Centro Hospitalar de Lisboa Central, Lisboa, Portugal; d Vivantes Humboldt-Klinikum, Berlin, Germany; e Vivantes Hospital Spandau, Berlin, e Hospital, AP-HP, University of Versailles Saint Germany; f Raymond-Poincar Quentin, Garches, France; g Merz Pharmaceuticals GmbH, Frankfurt am Main, Germany; h Hannover Medical School, Hannover, Germany; i Icahn School of Medicine at Mount Sinai, New York, NY, USA * Corresponding author: Gailtal-Klinik, Radniger Strasse 12, A-9620 Hermagor, Austria. E-mail address:[email protected].
Introduction and objectives: The safety and efficacy of botulinum toxin treatment for limb spasticity are well established. However, reports on the effect of treatment on quality of life (QoL) are inconclusive. Here we report QoL outcomes following incobotulinumtoxinA (Xeomin) treatment of upper and lower limb spasticity in the TOWER study. Methods: The open-label TOWER study (NCT01603459) assessed the safety and efficacy of escalating incobotulinumtoxinA doses (400 U, 600 U, and 600-800 U) in patients (ages 18-80 years) with upper and lower limb spasticity on the same body side due to stroke or other cerebral causes. QoL was assessed using the EQ-5D questionnaire, which includes 5 dimensions (mobility, self-care, usual activities, pain/discomfort, anxiety/depression), and a visual analog scale (VAS) on which patients rate their current state of health from 0 (worst) to 100 (best). Results: Patients (N¼155) were enrolled and received incobotulinumtoxinA. For all EQ-5D dimensions, the proportion of patients with improvements from study baseline to end of injection cycle (IC) 3 was higher than the proportion with worsening: mobility, 11.4% vs 2.9%; self-care, 21.4% vs 7.1%; usual activities, 24.3% vs 5.0%; pain/discomfort, 30.7% vs 9.3%; anxiety/depression, 25.0% vs 9.3%. The mean (standard deviation; SD) change in EQ-5D VAS score from study baseline to 4 weeks post-treatment was 6.7 (14.1) points, 9.6 (16.3) points, and 8.6 (17.0) points in IC1, IC2, and IC3, respectively (P<0.0001 for all). The mean (SD) EQ-5D VAS score improved from 59.9 (18.9) at baseline to 70.5 (16.7) at the end of IC3, corresponding to a mean (SD) change of 10.5 (17.5) points (P<0.0001).
1
DD and DS contributed equally to this abstract.
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