- Email: [email protected]
134. Potency assay for botulinum neurotoxin type A based on neuronal cells as a replacement for the mouse bioassay
Abstracts / Toxicon 93 (2015) S2eS67
R21AI101539, R01AI104579-01; and by cooperative agreement U01 AI056493 (PI: James Marks); and by the Centers for Disease Control under grants 00HCUGDH-2011-00996 and 200-2009-30597 (PI: James Marks); and by the Defense Threat Reduction Agency under contract HDTRA1-07C-0030 (PI: James Marks). Keywords: Anti-toxin; BoNT; ELISA; FACS; Human antibody; KinExA; Phage display; Yeast display 132. INSIGHTS INTO THE GLYCOSYLATION-STATUS DEPENDENT INTERACTION OF SV2C WITH BOTULINUM NEUROTOXIN TYPE A Stefan Mahrhold a,*, Jasmin Weisemann a, Thomas Binz b, Andreas Rummel a a Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany; b Institut für Biochemie, Medizinische Hochschule Hannover, Hannover, Germany *Corresponding author: Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail address: [email protected]
Introduction and Objectives: The botulinum neurotoxin (BoNT) serotypes A through G are among the most poisonous substances known. Their extreme potency is generated by the specific interaction of BoNTs with the neuronal plasma membrane, among other factors. It was demonstrated that BoNT serotypes A and E exploit binding to the synaptic vesicle (glyco-) protein 2 (SV2) on neuronal membranes to enter peripheral motoneurons. SV2 isoform A (SV2A) and C (SV2C) contain 3 and 5 putative N-glycosylation (PNG) sites, respectively, in the large luminal domain. It was shown that BoNT/E strictly requires glycosylation of asparagine 573 (N573) of SV2A to be able to bind and enter the target cell. Also, the binding of BoNT/ A to SV2A might benefit from the presence of this N-glycan. In this study, we addressed the question of whether the interaction of BoNT/A with SV2C, which is characterized by the highest direct protein-protein affinity, is likewise affected by the glycosylation of the homologous asparagine 559 (N559) or the neighboring N565 of human SV2C. Methods: Several mutations were introduced into human SV2C to prevent the glycosylation of N559 and N565, respectively. To be able to distinguish between effects elicited by the removal of the N-glycan and those caused by the mutations themselves, the corresponding proteins were expressed as soluble Fc-fusion proteins either in human embryonic kidney (HEK) cells to obtain glycosylated protein (gSV2C-Fc) or in Escherichia coli to yield completely nonglycosylated protein (SV2C-Fc). Purified SV2C fusion proteins were immobilized on protein G-sepharose beads and used as bait in pull-down experiments to test their ability to bind to full-length BoNT/A or BoNT/A- binding domain (HCA). Bound proteins were visualized by SDSPAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Coomassie Brilliant Blue staining, and the binding of mutants was compared with wild-type proteins, which were produced by the same expression system. Results: Sufficient amounts of soluble Fc-fusion proteins were obtained from culture supernatants of transiently transfected HEK293T cells (gSV2C-Fc) as well as from lysates of correspondingly transformed E. coli (SV2C-Fc). Mutation of the fourth (N559Q) or fifth (N565Q) PNG site of the human SV2C luminal domain resulted in an increased mobility of the gSV2C-Fc mutants in SDS-PAGE compared with wild-type, indicating that both sites are N-glycosylated. The double mutant gSV2C-Fc N559Q/N565Q showed an even stronger increase in mobility, confirming the simultaneous N-glycosylation of both sites in HEK cells. Wild-type gSV2C-Fc and SV2C-Fc showed a robust binding to BoNT/A and HCA, respectively. Mutant gSV2C-Fc N565Q exhibited binding to HCA indistinguishable from wildtype gSV2C-Fc, indicating that neither the N-glycan at N565 nor the amino acid N565 contribute to binding. In contrast, mutants gSV2C-Fc N559Q and SV2C-Fc N559Q both displayed a strong reduction in binding to BoNT/A and HCA, respectively. Hence, this result does not permit discrimination between effects caused by the missing N-glycan at N559 and effects generated by the amino acid exchange N559Q. However, mutant SV2C-Fc N559A showed no effect on binding to BoNT/A, whereas gSV2C-Fc N559A exhibited a strong reduction in binding to HCA, demonstrating that the presence of an N-glycan at N559 indeed facilitates binding to HCA. Conclusions: We show that the interaction of BoNT/A to SV2C is aided by the presence of an N-glycan at N559. This resembles the situation for the BoNT/E-SV2A interaction, which is also facilitated by the presence of an Nglycan at the homologous residue N573. The co-crystal structure of the
S41
human SV2C luminal domain in complex with HCA demonstrates that N559 is located at the binding interface between SV2C and HCA. An Nglycan attached to this residue most likely extends into the recently identified SV2 binding site of BoNT/A thereby explaining the increased affinity of gSV2C to BoNT/A. Additionally, we demonstrate the successful expression of soluble and glycosylated SV2 peptides with the help of transiently transfected HEK cells. Keywords: Botulinum neurotoxin type A; N-glycosylation; Receptor interaction; SV2C; Synaptic vesicle (glyco-) protein 2C 133. EFFECTS OF BOTULINUM TOXIN ON TREATMENT FOR SIALORRHEA IN PATIENTS WITH CEREBRAL PALSY Claudia Marques Santa Rosa Malcher, Nicolau Conte ^ nio Rebelo Pontes, Jo~ ^s Helder Anto ao Amaury France Joaquim Bentes Gomes da Silva, Miguel Akkari* ~o Paulo, Sao Paulo, Brazil rdia de Sa Santa Casa de Miserico
Neto, Brito,
~o Paulo, Street Dr. Cesare *Corresponding author: Santa Casa de Misericord de Sa Motta Jr, 112 Santa Cecilia, Sao Paulo, 01221-900, Brazil. E-mail address: miguel. [email protected]
Introduction and Objectives: In patients with cerebral palsy, sialorrhea is a common problem leading to clinical and functional complications such as aspiration, skin breakdown, bad odor, infections, and impairment in social functioning, causing embarrassment and isolation. Because of these problems, it is necessary to find a treatment that has positive effects on the clinical manifestations of this disease, with decreased complications. Methods: An interventional, longitudinal, and prospective study was developed with 4 assessments at 0, 15, 60, and 90 days and corresponding to the times T0, T1, T2, and T3, for use in patients with cerebral palsy who were treated with botulinum neurotoxin type A, as seen in the Institute of , Brazil, from February 1 to July 31, 2013. Toxin Botulinum Application, Para This work was approved by Human Research Ethics Committee. CAAE: 10718312.10000.0018. Results: Of the 20 patients evaluated, 55% were male and 45% were female; ages were from 5 to 29 years (mean, 10.9; median, 9 years). Of the 4 scales used to evaluate decrease in drooling (frequency, severity, overproduction, number of bibs), positive results (P<0.05) were seen at T1 for overproduction and number of bibs, and best results at T2 and T3 in all scales, using Kruskal-Wallis and Dunn’s statistical tests post hoc. The most common complications reported were thick saliva (30%), pain and edema (25%), and redness (10%) of slight intensity at application site. The difficulties were its temporary effect, with a need for new application in more than half (65%) of patients at 90 days. Conclusions: This treatment was an effective alternative for control of drooling after application at 15, 60, and 90 days in 95% of cases. Because of the good clinical results, this treatment may be an effective and safe alternative in patients with cerebral palsy. Keywords: Botulinum toxin; Cerebral palsy; Drooling; Therapy
134. POTENCY ASSAY FOR BOTULINUM NEUROTOXIN TYPE A BASED ON NEURONAL CELLS AS A REPLACEMENT FOR THE MOUSE BIOASSAY Gerd Mander a, Cornelia Bruenn a, Claudia Jatzke a, Karl-Heinz Eisele a, Harold V. Taylor a, Sabine Pellett b, Eric A. Johnson b, Klaus Fink a, * a Merz Pharmaceuticals, Frankfurt, Germany; b Department of Bacteriology, Botulinum Toxins Laboratory, University of Wisconsin, WI, USA. *Corresponding author: Eckenheimer Landstr. 100, 60318 Frankfurt, Germany. E-mail address: klaus.fi[email protected]
The potency of botulinum neurotoxin drug products has been measured for 30 years using the mouse lethal dose, 50% (LD50) bioassay as described in the European Pharmacopoeia and the US Pharmacopoeial Convention. The LD50 assay has been questioned due to the immanent stress for the animals. In vivo local paralysis assays such as the mouse flaccid paralysis or rat grip strength assays, ex vivo assays such as the mouse hemidiaphragm assay, and in vitro assays based on primary neurons were sufficiently sensitive but could not be validated according to International Conference on Harmonization (ICH) guidelines. Very
S42
Abstracts / Toxicon 93 (2015) S2eS67
few neuroblastoma cell lines turned out to be sensitive enough (Fernandez-Salas 2012). We used human induced, stem cell‒derived neuronal cells (Whitemarsh 2012) to design a potency assay for BoNT/A drug product Xeomin/Bocouture(incobotulinumtoxinA). This assay reflects all BoNT mode-of-action steps: binding, internalization, translocation, and proteolytic activity. Human-induced pluripotent stem cells differentiated into neuronal cells were obtained from Cellular Dynamics International, plated, and grown in 96-well plates. The cultured neuronal cells were exposed to incobotulinumtoxinA at 1.7E-12 mol/L- 1.3E-18 mol/L for 24 to 96 hours. The medium was removed, cells were permeabilized, and antibodies specific for the BoNT/A-cleaved synaptosomal-associated protein of 25kDa (SNAP)-25197 neoepitope and, for normalization, total SNAP-25 were used for detection. The assay determines biological activity at an half maximal effective concentration (EC50) of 3x1015 M and is capable of measuring biological activity in incobotulinumtoxinA. The BoNT/A potency assay based on human induced pluripotent stem cell‒derived neuronal cells fully replaces the mouse lethal dose 50% (LD50) bioassay for incobotulinumtoxinA. Advantages of the assay are that it is based on noncancerous cells, normalized for total SNAP-25, more sensitive and less variable than the mouse bioassay, specific for BoNT/A, and validated according to the ICHQ2 guideline. References ndez-Salas E, Wang J, Molina Y, Nelson JB, Jacky BPS, Aoki KR. BotuFerna linum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay. PLoS One. 2012;7(11):e49516. doi:10.1371/journal. pone.0049516. Whitemarsh RCM, Strathman MJ, Chase LG, et al. Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection. Toxicol Sci. 2012;126(2): 426-435. Keywords: Cell-based potency assay; Mouse LD50 bioassay 135. BOTULINUM TOXIN DYSTONIA OUTCOMES: COMPARING PRIMARY AND SECONDARY BLEPHAROSPASM PATIENTS Daniel Martinez-Ramirez*, Juan C. Giugni, Erin Hastings, Aparna Wagle-Shukla, Irene Malaty, Michael S. Okun, Ramon Rodriguez Center for Movement Disorders and Neurorestoration, University of Florida, Gainesville, FL, USA *Corresponding author: Center for Movement Disorders and Neurorestoration, Department of Neurology, University of Florida, 3450 Hull Road, 4th Floor, Gainesville, FL 32607, USA. E-mail address: [email protected]fl.edu
Introduction and Objectives: Benign essential blepharospasm (BEB) is a dystonia that impairs quality of life and is classified as primary or secondary. Botulinum neurotoxin (BoNT) is considered an effective primary therapy; however, no studies have compared outcomes in patients with primary and secondary BEB. We compared responses in both groups and examined factors affecting the outcomes. Methods: A retrospective chart review was conducted. First and last procedure, duration and degree of benefit, and side effects were all recorded. Treatment outcomes were analyzed using a self-report scale for the duration of benefit (in weeks) and the Clinical Global Impression Scale. Results: Sixty-four patients were included. Primary BEB was more common (64% vs 36%). Symptom duration was longer in primary than in secondary BEB (14.2 years, [standard deviation] SD: 11.9 vs 6.2 years, SD: 4.7; P¼0.003). There was male predominance (74% vs 32%) in the secondary group. There were no differences between the 2 cohorts in duration of benefit of BoNT treatment. Although peak dose benefit was more commonly reported as “very much improved” in secondary BEB patients, the difference was not statistically significant (P¼0.13). Higher BoNT dosages were required over time in both groups. Ptosis (8%) and diplopia (6%) were the most commonly reported side effects. Conclusions: BoNT injections were effective and conferred similar benefits in all BEB patients whether primary or secondary. The duration of benefit in this study population was a little shorter than reported in other clinical trials. Prospective trials are needed to confirm these findings Keywords: Blepharospasm; Botulinum toxin; Dystonia
136. ANTINOCICEPTIVE ACTIVITY OF BOTULINUM TOXIN TYPE A IN THE RAT TRIGEMINAL REGION Ivica Matak*, Boris Filipovi c, Visnja Drinovac, Lidija Bach-Rojecky, Zdravko Lackovi c University of Zagreb School of Medicine, Zagreb, Croatia *Corresponding author: Salata 11, 10000 Zagreb, Croatia. E-mail address: ivica. [email protected]
Meta-analyses suggest that botulinum toxin type A (BoNT/A) is effective for treatment of chronic migraine, chronic daily headache, and trigeminal neuralgia. It is also suggested that BoNT/A is effective in medication overuse headache. However, generally, little is known about the mechanisms of its action on headache and craniofacial pain. Thus, we investigated the BoNT/A antinociceptive action in the rat trigeminal region. BoNT/A did not alter the normal response to innocuous and noxious mechanical stimuli, but it reduced phase II hyperalgesia in an orofacial formalin test and bilateral allodynia in a model of trigeminal neuropathy. In both pain models, BoNT/A reduced the neurogenic inflammation (plasma protein extravasation) in the dura mater. Its action on pain and dural inflammation was prevented by colchicine, an intra-ganglionic axonal transport blocker. After facial injection, BoNT/A-truncated synaptosomal-associated protein 25 kDa (SNAP-25) was found in the trigeminal nucleus caudalis, trigeminal nucleus oralis, and cervical spinal cord. BoNT/A protease activity was localized in capsaicin-sensitive central afferent terminals. Denervation of capsaicin-sensitive neurons prevented the BoNT/A antinociceptive action. BoNT/A reduced the painevoked neuronal activation in the trigeminal nucleus caudalis, locus coeruleus, and periaqueductal gray. The toxin’s antinociceptive action on the orofacial formalin test was blocked by naltrexone, an opioid antagonist. The effect of BoNT/A on pain hypersensitivity in the trigeminal region is dependent on axonal transport of the toxin via the sensory neurons from the periphery to the central nervous system. Prevention of neurogenic peptide release in the cranial meninges might be associated with BoNT/A anti-migraine efficacy. Activity in capsaicin-sensitive primary afferents is in line with BoNT/A selectivity for allodynia and hyperalgesia. BoNT/A prevents pain-evoked neuronal activity in migraine-associated sensory regions. Its activity on pain in the trigeminal region involves activation of the endogenous opioid system. Keywords: Axonal transport; Botulinum toxin type A; Capsaicin-sensitive neurons; Dural neurogenic inflammation; Synaptosomal-associated protein 25; Trigeminal system 137. ONABOTULINUMTOXINA PROPHYLAXIS IN CHRONIC MIGRAINE UTILIZATION AND PATIENT CHARACTERISTICS: OBSERVATIONAL STUDY IN THE EUROPEAN UNION Manjit Matharu a, Julio Pascual Gomez b, Ingela Nilsson Remahl c, Dawn Odom d, Lia Gutierrez e, Elizabeth Andrews d, Joan Largent f, *, Catherine Johannes g a National Hospital for Neurology and Neurosurgery, London, UK; b Hospital Universitario Central de Asturias (HUCA) and INEUROPA, Oviedo, Spain; c Karolinska University Hospital Huddinge, Stockholm, Sweden; d RTI Health Solutions, Research Triangle Park, NC, USA; e RTI Health Solutions, Travesera de Gracia, Barcelona, Spain; f Allergan, Irvine, CA, USA; g RTI Health Solutions, Waltham, MA, USA *Corresponding author: 2525 Dupont Drive, Irvine, CA 92612, USA. E-mail address: [email protected]
Introduction and Objectives: Available treatments for chronic migraine (CM) include abortive migraine drugs, oral migraine prophylaxis, and onabotuliumtoxinA (onabotA) prophylaxis. This study evaluates onabotA utilization for CM headache prophylaxis in clinical practice and describes practice, physician, and patient characteristics. Methods: This is a prospective, noninterventional, multinational European postauthorization study among CM adults treated with onabotA. Participating physicians recruited patients receiving treatment during routine care (September 2011 to February 2014). The study describes usage patterns and safety profile of onabotA. Baseline characteristics
R21AI101539, R01AI104579-01; and by cooperative agreement U01 AI056493 (PI: James Marks); and by the Centers for Disease Control under grants 00HCUGDH-2011-00996 and 200-2009-30597 (PI: James Marks); and by the Defense Threat Reduction Agency under contract HDTRA1-07C-0030 (PI: James Marks). Keywords: Anti-toxin; BoNT; ELISA; FACS; Human antibody; KinExA; Phage display; Yeast display 132. INSIGHTS INTO THE GLYCOSYLATION-STATUS DEPENDENT INTERACTION OF SV2C WITH BOTULINUM NEUROTOXIN TYPE A Stefan Mahrhold a,*, Jasmin Weisemann a, Thomas Binz b, Andreas Rummel a a Institut für Toxikologie, Medizinische Hochschule Hannover, Hannover, Germany; b Institut für Biochemie, Medizinische Hochschule Hannover, Hannover, Germany *Corresponding author: Carl-Neuberg-Str. 1, 30625 Hannover, Germany. E-mail address: [email protected]
Introduction and Objectives: The botulinum neurotoxin (BoNT) serotypes A through G are among the most poisonous substances known. Their extreme potency is generated by the specific interaction of BoNTs with the neuronal plasma membrane, among other factors. It was demonstrated that BoNT serotypes A and E exploit binding to the synaptic vesicle (glyco-) protein 2 (SV2) on neuronal membranes to enter peripheral motoneurons. SV2 isoform A (SV2A) and C (SV2C) contain 3 and 5 putative N-glycosylation (PNG) sites, respectively, in the large luminal domain. It was shown that BoNT/E strictly requires glycosylation of asparagine 573 (N573) of SV2A to be able to bind and enter the target cell. Also, the binding of BoNT/ A to SV2A might benefit from the presence of this N-glycan. In this study, we addressed the question of whether the interaction of BoNT/A with SV2C, which is characterized by the highest direct protein-protein affinity, is likewise affected by the glycosylation of the homologous asparagine 559 (N559) or the neighboring N565 of human SV2C. Methods: Several mutations were introduced into human SV2C to prevent the glycosylation of N559 and N565, respectively. To be able to distinguish between effects elicited by the removal of the N-glycan and those caused by the mutations themselves, the corresponding proteins were expressed as soluble Fc-fusion proteins either in human embryonic kidney (HEK) cells to obtain glycosylated protein (gSV2C-Fc) or in Escherichia coli to yield completely nonglycosylated protein (SV2C-Fc). Purified SV2C fusion proteins were immobilized on protein G-sepharose beads and used as bait in pull-down experiments to test their ability to bind to full-length BoNT/A or BoNT/A- binding domain (HCA). Bound proteins were visualized by SDSPAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and Coomassie Brilliant Blue staining, and the binding of mutants was compared with wild-type proteins, which were produced by the same expression system. Results: Sufficient amounts of soluble Fc-fusion proteins were obtained from culture supernatants of transiently transfected HEK293T cells (gSV2C-Fc) as well as from lysates of correspondingly transformed E. coli (SV2C-Fc). Mutation of the fourth (N559Q) or fifth (N565Q) PNG site of the human SV2C luminal domain resulted in an increased mobility of the gSV2C-Fc mutants in SDS-PAGE compared with wild-type, indicating that both sites are N-glycosylated. The double mutant gSV2C-Fc N559Q/N565Q showed an even stronger increase in mobility, confirming the simultaneous N-glycosylation of both sites in HEK cells. Wild-type gSV2C-Fc and SV2C-Fc showed a robust binding to BoNT/A and HCA, respectively. Mutant gSV2C-Fc N565Q exhibited binding to HCA indistinguishable from wildtype gSV2C-Fc, indicating that neither the N-glycan at N565 nor the amino acid N565 contribute to binding. In contrast, mutants gSV2C-Fc N559Q and SV2C-Fc N559Q both displayed a strong reduction in binding to BoNT/A and HCA, respectively. Hence, this result does not permit discrimination between effects caused by the missing N-glycan at N559 and effects generated by the amino acid exchange N559Q. However, mutant SV2C-Fc N559A showed no effect on binding to BoNT/A, whereas gSV2C-Fc N559A exhibited a strong reduction in binding to HCA, demonstrating that the presence of an N-glycan at N559 indeed facilitates binding to HCA. Conclusions: We show that the interaction of BoNT/A to SV2C is aided by the presence of an N-glycan at N559. This resembles the situation for the BoNT/E-SV2A interaction, which is also facilitated by the presence of an Nglycan at the homologous residue N573. The co-crystal structure of the
S41
human SV2C luminal domain in complex with HCA demonstrates that N559 is located at the binding interface between SV2C and HCA. An Nglycan attached to this residue most likely extends into the recently identified SV2 binding site of BoNT/A thereby explaining the increased affinity of gSV2C to BoNT/A. Additionally, we demonstrate the successful expression of soluble and glycosylated SV2 peptides with the help of transiently transfected HEK cells. Keywords: Botulinum neurotoxin type A; N-glycosylation; Receptor interaction; SV2C; Synaptic vesicle (glyco-) protein 2C 133. EFFECTS OF BOTULINUM TOXIN ON TREATMENT FOR SIALORRHEA IN PATIENTS WITH CEREBRAL PALSY Claudia Marques Santa Rosa Malcher, Nicolau Conte ^ nio Rebelo Pontes, Jo~ ^s Helder Anto ao Amaury France Joaquim Bentes Gomes da Silva, Miguel Akkari* ~o Paulo, Sao Paulo, Brazil rdia de Sa Santa Casa de Miserico
Neto, Brito,
~o Paulo, Street Dr. Cesare *Corresponding author: Santa Casa de Misericord de Sa Motta Jr, 112 Santa Cecilia, Sao Paulo, 01221-900, Brazil. E-mail address: miguel. [email protected]
Introduction and Objectives: In patients with cerebral palsy, sialorrhea is a common problem leading to clinical and functional complications such as aspiration, skin breakdown, bad odor, infections, and impairment in social functioning, causing embarrassment and isolation. Because of these problems, it is necessary to find a treatment that has positive effects on the clinical manifestations of this disease, with decreased complications. Methods: An interventional, longitudinal, and prospective study was developed with 4 assessments at 0, 15, 60, and 90 days and corresponding to the times T0, T1, T2, and T3, for use in patients with cerebral palsy who were treated with botulinum neurotoxin type A, as seen in the Institute of , Brazil, from February 1 to July 31, 2013. Toxin Botulinum Application, Para This work was approved by Human Research Ethics Committee. CAAE: 10718312.10000.0018. Results: Of the 20 patients evaluated, 55% were male and 45% were female; ages were from 5 to 29 years (mean, 10.9; median, 9 years). Of the 4 scales used to evaluate decrease in drooling (frequency, severity, overproduction, number of bibs), positive results (P<0.05) were seen at T1 for overproduction and number of bibs, and best results at T2 and T3 in all scales, using Kruskal-Wallis and Dunn’s statistical tests post hoc. The most common complications reported were thick saliva (30%), pain and edema (25%), and redness (10%) of slight intensity at application site. The difficulties were its temporary effect, with a need for new application in more than half (65%) of patients at 90 days. Conclusions: This treatment was an effective alternative for control of drooling after application at 15, 60, and 90 days in 95% of cases. Because of the good clinical results, this treatment may be an effective and safe alternative in patients with cerebral palsy. Keywords: Botulinum toxin; Cerebral palsy; Drooling; Therapy
134. POTENCY ASSAY FOR BOTULINUM NEUROTOXIN TYPE A BASED ON NEURONAL CELLS AS A REPLACEMENT FOR THE MOUSE BIOASSAY Gerd Mander a, Cornelia Bruenn a, Claudia Jatzke a, Karl-Heinz Eisele a, Harold V. Taylor a, Sabine Pellett b, Eric A. Johnson b, Klaus Fink a, * a Merz Pharmaceuticals, Frankfurt, Germany; b Department of Bacteriology, Botulinum Toxins Laboratory, University of Wisconsin, WI, USA. *Corresponding author: Eckenheimer Landstr. 100, 60318 Frankfurt, Germany. E-mail address: klaus.fi[email protected]
The potency of botulinum neurotoxin drug products has been measured for 30 years using the mouse lethal dose, 50% (LD50) bioassay as described in the European Pharmacopoeia and the US Pharmacopoeial Convention. The LD50 assay has been questioned due to the immanent stress for the animals. In vivo local paralysis assays such as the mouse flaccid paralysis or rat grip strength assays, ex vivo assays such as the mouse hemidiaphragm assay, and in vitro assays based on primary neurons were sufficiently sensitive but could not be validated according to International Conference on Harmonization (ICH) guidelines. Very
S42
Abstracts / Toxicon 93 (2015) S2eS67
few neuroblastoma cell lines turned out to be sensitive enough (Fernandez-Salas 2012). We used human induced, stem cell‒derived neuronal cells (Whitemarsh 2012) to design a potency assay for BoNT/A drug product Xeomin/Bocouture(incobotulinumtoxinA). This assay reflects all BoNT mode-of-action steps: binding, internalization, translocation, and proteolytic activity. Human-induced pluripotent stem cells differentiated into neuronal cells were obtained from Cellular Dynamics International, plated, and grown in 96-well plates. The cultured neuronal cells were exposed to incobotulinumtoxinA at 1.7E-12 mol/L- 1.3E-18 mol/L for 24 to 96 hours. The medium was removed, cells were permeabilized, and antibodies specific for the BoNT/A-cleaved synaptosomal-associated protein of 25kDa (SNAP)-25197 neoepitope and, for normalization, total SNAP-25 were used for detection. The assay determines biological activity at an half maximal effective concentration (EC50) of 3x1015 M and is capable of measuring biological activity in incobotulinumtoxinA. The BoNT/A potency assay based on human induced pluripotent stem cell‒derived neuronal cells fully replaces the mouse lethal dose 50% (LD50) bioassay for incobotulinumtoxinA. Advantages of the assay are that it is based on noncancerous cells, normalized for total SNAP-25, more sensitive and less variable than the mouse bioassay, specific for BoNT/A, and validated according to the ICHQ2 guideline. References ndez-Salas E, Wang J, Molina Y, Nelson JB, Jacky BPS, Aoki KR. BotuFerna linum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay. PLoS One. 2012;7(11):e49516. doi:10.1371/journal. pone.0049516. Whitemarsh RCM, Strathman MJ, Chase LG, et al. Novel application of human neurons derived from induced pluripotent stem cells for highly sensitive botulinum neurotoxin detection. Toxicol Sci. 2012;126(2): 426-435. Keywords: Cell-based potency assay; Mouse LD50 bioassay 135. BOTULINUM TOXIN DYSTONIA OUTCOMES: COMPARING PRIMARY AND SECONDARY BLEPHAROSPASM PATIENTS Daniel Martinez-Ramirez*, Juan C. Giugni, Erin Hastings, Aparna Wagle-Shukla, Irene Malaty, Michael S. Okun, Ramon Rodriguez Center for Movement Disorders and Neurorestoration, University of Florida, Gainesville, FL, USA *Corresponding author: Center for Movement Disorders and Neurorestoration, Department of Neurology, University of Florida, 3450 Hull Road, 4th Floor, Gainesville, FL 32607, USA. E-mail address: [email protected]fl.edu
Introduction and Objectives: Benign essential blepharospasm (BEB) is a dystonia that impairs quality of life and is classified as primary or secondary. Botulinum neurotoxin (BoNT) is considered an effective primary therapy; however, no studies have compared outcomes in patients with primary and secondary BEB. We compared responses in both groups and examined factors affecting the outcomes. Methods: A retrospective chart review was conducted. First and last procedure, duration and degree of benefit, and side effects were all recorded. Treatment outcomes were analyzed using a self-report scale for the duration of benefit (in weeks) and the Clinical Global Impression Scale. Results: Sixty-four patients were included. Primary BEB was more common (64% vs 36%). Symptom duration was longer in primary than in secondary BEB (14.2 years, [standard deviation] SD: 11.9 vs 6.2 years, SD: 4.7; P¼0.003). There was male predominance (74% vs 32%) in the secondary group. There were no differences between the 2 cohorts in duration of benefit of BoNT treatment. Although peak dose benefit was more commonly reported as “very much improved” in secondary BEB patients, the difference was not statistically significant (P¼0.13). Higher BoNT dosages were required over time in both groups. Ptosis (8%) and diplopia (6%) were the most commonly reported side effects. Conclusions: BoNT injections were effective and conferred similar benefits in all BEB patients whether primary or secondary. The duration of benefit in this study population was a little shorter than reported in other clinical trials. Prospective trials are needed to confirm these findings Keywords: Blepharospasm; Botulinum toxin; Dystonia
136. ANTINOCICEPTIVE ACTIVITY OF BOTULINUM TOXIN TYPE A IN THE RAT TRIGEMINAL REGION Ivica Matak*, Boris Filipovi c, Visnja Drinovac, Lidija Bach-Rojecky, Zdravko Lackovi c University of Zagreb School of Medicine, Zagreb, Croatia *Corresponding author: Salata 11, 10000 Zagreb, Croatia. E-mail address: ivica. [email protected]
Meta-analyses suggest that botulinum toxin type A (BoNT/A) is effective for treatment of chronic migraine, chronic daily headache, and trigeminal neuralgia. It is also suggested that BoNT/A is effective in medication overuse headache. However, generally, little is known about the mechanisms of its action on headache and craniofacial pain. Thus, we investigated the BoNT/A antinociceptive action in the rat trigeminal region. BoNT/A did not alter the normal response to innocuous and noxious mechanical stimuli, but it reduced phase II hyperalgesia in an orofacial formalin test and bilateral allodynia in a model of trigeminal neuropathy. In both pain models, BoNT/A reduced the neurogenic inflammation (plasma protein extravasation) in the dura mater. Its action on pain and dural inflammation was prevented by colchicine, an intra-ganglionic axonal transport blocker. After facial injection, BoNT/A-truncated synaptosomal-associated protein 25 kDa (SNAP-25) was found in the trigeminal nucleus caudalis, trigeminal nucleus oralis, and cervical spinal cord. BoNT/A protease activity was localized in capsaicin-sensitive central afferent terminals. Denervation of capsaicin-sensitive neurons prevented the BoNT/A antinociceptive action. BoNT/A reduced the painevoked neuronal activation in the trigeminal nucleus caudalis, locus coeruleus, and periaqueductal gray. The toxin’s antinociceptive action on the orofacial formalin test was blocked by naltrexone, an opioid antagonist. The effect of BoNT/A on pain hypersensitivity in the trigeminal region is dependent on axonal transport of the toxin via the sensory neurons from the periphery to the central nervous system. Prevention of neurogenic peptide release in the cranial meninges might be associated with BoNT/A anti-migraine efficacy. Activity in capsaicin-sensitive primary afferents is in line with BoNT/A selectivity for allodynia and hyperalgesia. BoNT/A prevents pain-evoked neuronal activity in migraine-associated sensory regions. Its activity on pain in the trigeminal region involves activation of the endogenous opioid system. Keywords: Axonal transport; Botulinum toxin type A; Capsaicin-sensitive neurons; Dural neurogenic inflammation; Synaptosomal-associated protein 25; Trigeminal system 137. ONABOTULINUMTOXINA PROPHYLAXIS IN CHRONIC MIGRAINE UTILIZATION AND PATIENT CHARACTERISTICS: OBSERVATIONAL STUDY IN THE EUROPEAN UNION Manjit Matharu a, Julio Pascual Gomez b, Ingela Nilsson Remahl c, Dawn Odom d, Lia Gutierrez e, Elizabeth Andrews d, Joan Largent f, *, Catherine Johannes g a National Hospital for Neurology and Neurosurgery, London, UK; b Hospital Universitario Central de Asturias (HUCA) and INEUROPA, Oviedo, Spain; c Karolinska University Hospital Huddinge, Stockholm, Sweden; d RTI Health Solutions, Research Triangle Park, NC, USA; e RTI Health Solutions, Travesera de Gracia, Barcelona, Spain; f Allergan, Irvine, CA, USA; g RTI Health Solutions, Waltham, MA, USA *Corresponding author: 2525 Dupont Drive, Irvine, CA 92612, USA. E-mail address: [email protected]
Introduction and Objectives: Available treatments for chronic migraine (CM) include abortive migraine drugs, oral migraine prophylaxis, and onabotuliumtoxinA (onabotA) prophylaxis. This study evaluates onabotA utilization for CM headache prophylaxis in clinical practice and describes practice, physician, and patient characteristics. Methods: This is a prospective, noninterventional, multinational European postauthorization study among CM adults treated with onabotA. Participating physicians recruited patients receiving treatment during routine care (September 2011 to February 2014). The study describes usage patterns and safety profile of onabotA. Baseline characteristics