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Augmentation of the catalytic activity of Botulinum neurotoxin type B does not result in increased potency in physiologic systems
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Abstracts / Toxicon 123 (2016) S2eS90
in the adductor longus muscle (2 sites) under EMG guidance. The primary efficacy variable of the study was HHS score at week 4 posttreatment. The secondary variables were VAS score, MRC value, and SF-36 score at weeks 2, 4, and 12 posttreatment; and HHS score at weeks 2 and 12 posttreatment. Results: The Statistical Package for Social Sciences Software 13.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis. A P value less than 0.05 was used for rejection of the null hypothesis.The significance of the differences in KL grade was evaluated using the Mann-Whitney U-test. For all other parameters, the Mann-Whitney U test was used to assess the significance of differences between the groups within each time point. The significance of the differences over time within either group was assessed using a Friedman test followed by a Bonferroni-corrected Wilcoxon paired signed-rank test, where appropriate. The DG group showed a significant difference compared to the PG group at 4 weeks for the HHS and VAS. There were no AED or significant changes. Values for weight, height, age, and KL, MRC, and SF-36 (both physical and mental components) scores were similar between the groups at baseline with no significant differences. Values for the HHS, VAS, MRC, and SF-36 (both physical and mental components) are summarized in Table 1. Conclusions: In HOA, abobotulinumtoxinA injection in muscles involved in hip movement enables a substantial and immediate restoration of the best functional activity of the hip with remarkable benefits in the mobility of the joint and a reduction of pain. Therefore, this therapy is useful in delaying THA or improving mobility and pain for patients awaiting surgery. Keywords: AbobotulinumtoxinA; Coxarthrosis; Hip; Pain References 1. Marchini C, Acler M, Bolognari MA, et al. Efficacy of botulinum toxin type A treatment of functional impairment of degenerative hip joint: preliminary results. J Rehabil Med. 2010;42(7):691-693. 2. Harris WH. Traumatic arthritis of the hip after dislocation and acetabular fractures: treatment by mold arthroplasty. An end-result study using a new method of result evaluation. J Bone Joint Surg Am. 1969;51(4):737-755. 3. Bellamy N. WOMAC Osteoarthritis Index: A Users Guide. London, Ontario, Canada: University of Western Ontario;1995. 65. AUGMENTATION OF THE CATALYTIC ACTIVITY OF BOTULINUM NEUROTOXIN TYPE B DOES NOT RESULT IN INCREASED POTENCY IN PHYSIOLOGIC SYSTEMS Mark Elliott a,*, Paul Farrow a, Christine Favre-Guilmard b, Fraser Hornby a, Sai Man Liu a, Jacquie Maignel-Ludop b, Sandra Marlin a, Imran Mir a, Shilpa Palan a, Mikhail Kalinichev b, Matthew Beard a, Johannes Krupp b. a Ipsen Bioinnovation, Abingdon, Oxfordshire, UK; b Ipsen Innovation, Les Ulis, France * Corresponding author: Ipsen Bioinnovation, 102 Park Drive, Milton Park, Abingdon, OX14 4RY, UK. E-mail address:[email protected].
Introduction: Botulinum neurotoxin serotype B (BoNT/B) is available as a marketed product; however, high doses are required to reach an efficacy similar to that of products containing BoNT/A. Higher doses of BoNT/B have been correlated with an increased incidence of neutralizing antibody production, which is part of the reason for the limited use of the BoNT/B product in the clinic. Recently, a mutation in the light chain of BoNT/B was described that increases the catalytic activity of the isolated BoNT/B light chain in biochemical assays (Guo 2013). In this study, we have produced a full-length recombinant BoNT/B toxin incorporating this mutation and assessed its activity in several biological systems.
Methods: The coding sequence for BoNT/B1 was cloned and expressed in Escherichia coli. The point mutation S201P described by Guo et al was introduced to create the mutated BoNT/B1(S201P). Both mutated and natural sequence toxins were purified and activated by the protease Lys-C. Purified, activated BoNT/B was characterized in cell-free cleavage assays, several cell-based assays, isolated tissue assays, and in the mouse Digit Abduction Score (DAS) test. Results: BoNT/B1(S201P) cleaved VAMP2 in a cell-free assay with higher activity than BoNT/B1, confirming the results of Guo and colleagues. However, there was no significant difference between these two toxins in any of the cell-based assays. Likewise, there was no difference in the inhibition of muscle activity in either of the tissue assays. Finally, in vivo mouse DAS scores as well as in vivo safety parameters were not different between the 2 toxins. Conclusions: Although BoNT/B1(S201P) has a higher catalytic activity than BoNT/B1 in a cell-free system, this does not translate into higher potency in complex biological systems. This probably indicates that the catalytic step is not the rate-limiting step for BoNT/B to reach efficacy. Funding: Ipsen Keywords: BoNT/B; Light chain; Recombinant; VAMP-2 Reference Guo J, Pan X, Zhao Y, Chen S. Engineering clostridia neurotoxins with elevated catalytic activity. Toxicon. 2013;74:158-166. 66. MUSCLE SELECTION FOR ONABOTULINUMTOXINA IN POSTSTROKE LOWER LIMB SPASTICITY INFLUENCES OUTCOME: RESULTS FROM A DOUBLE-BLIND, PLACEBO-CONTROLLED PHASE 3 CLINICAL TRIAL Alberto Esquenazi a, *, Carolyn Geis b, Theodore H. Wein c, Anthony B. Ward d, Chengcheng Liu e, Rozalina Dimitrova f. a MossRehab Gait and Motion Analysis Laboratory, Elkins Park, PA, USA; b Department of Rehabilitation Medicine, Brooks Rehabilitation Physician Group, Daytona Beach, FL, USA; c McGill University, Department of Neurology and Neurosurgery, Montreal General Hospital, Montreal, Quebec, Canada; d North Staffordshire Rehabilitation Centre, Staffordshire University, Stoke on Trent, UK; e Biostatistics, Allergan plc, Bridgewater, NJ, USA; f Neurology and Pain Clinical Development, Allergan plc, Irvine, CA, USA * Corresponding author: MossRehab Gait and Motion Analysis Laboratory, 60 Township Line Road, Elkins Park, PA 19207, USA. E-mail address:aesquena@einstein. edu.
Introduction and Objectives: Injector preference appears to be the main determinant of muscle selection pattern in the treatment of upper-limb spasticity (Baguley 2011). The optimal muscle selection pattern for treatment of poststroke lower limb spasticity (PSLLS) is not yet established. This analysis of the REFLEX study aims to identify an optimal muscle selection pattern for onabotulinumtoxinA (onabotA) injection for the treatment of PSLLS. Methods: The REFLEX study was a phase 3, placebo-controlled study in 60 international centers. Adults with PSLLS (Modified Ashworth Scale [MAS] 3 in the ankle plantar flexors) were eligible for inclusion. During the 12week double-blind phase, patients were randomly assigned to onabotA (300 U into ankle flexors, ie, gastrocnemius, soleus, and tibialis posterior and, optionally, 100 U total into flexor digitorum longus [FDL], flexor digitorum brevis, flexor hallucis longus [FHL], extensor hallucis longus, and/or rectus femoris muscles) or placebo. The primary endpoint, MAS change from baseline, and a secondary endpoint, physician-assessed Clinical Global Impression of Change (CGI), were each reported as the average score of weeks 4 and 6 (the time frame for pharmacologic effect).
Abstracts / Toxicon 123 (2016) S2eS90
in the adductor longus muscle (2 sites) under EMG guidance. The primary efficacy variable of the study was HHS score at week 4 posttreatment. The secondary variables were VAS score, MRC value, and SF-36 score at weeks 2, 4, and 12 posttreatment; and HHS score at weeks 2 and 12 posttreatment. Results: The Statistical Package for Social Sciences Software 13.0 (SPSS Inc., Chicago, IL, USA) was used for data analysis. A P value less than 0.05 was used for rejection of the null hypothesis.The significance of the differences in KL grade was evaluated using the Mann-Whitney U-test. For all other parameters, the Mann-Whitney U test was used to assess the significance of differences between the groups within each time point. The significance of the differences over time within either group was assessed using a Friedman test followed by a Bonferroni-corrected Wilcoxon paired signed-rank test, where appropriate. The DG group showed a significant difference compared to the PG group at 4 weeks for the HHS and VAS. There were no AED or significant changes. Values for weight, height, age, and KL, MRC, and SF-36 (both physical and mental components) scores were similar between the groups at baseline with no significant differences. Values for the HHS, VAS, MRC, and SF-36 (both physical and mental components) are summarized in Table 1. Conclusions: In HOA, abobotulinumtoxinA injection in muscles involved in hip movement enables a substantial and immediate restoration of the best functional activity of the hip with remarkable benefits in the mobility of the joint and a reduction of pain. Therefore, this therapy is useful in delaying THA or improving mobility and pain for patients awaiting surgery. Keywords: AbobotulinumtoxinA; Coxarthrosis; Hip; Pain References 1. Marchini C, Acler M, Bolognari MA, et al. Efficacy of botulinum toxin type A treatment of functional impairment of degenerative hip joint: preliminary results. J Rehabil Med. 2010;42(7):691-693. 2. Harris WH. Traumatic arthritis of the hip after dislocation and acetabular fractures: treatment by mold arthroplasty. An end-result study using a new method of result evaluation. J Bone Joint Surg Am. 1969;51(4):737-755. 3. Bellamy N. WOMAC Osteoarthritis Index: A Users Guide. London, Ontario, Canada: University of Western Ontario;1995. 65. AUGMENTATION OF THE CATALYTIC ACTIVITY OF BOTULINUM NEUROTOXIN TYPE B DOES NOT RESULT IN INCREASED POTENCY IN PHYSIOLOGIC SYSTEMS Mark Elliott a,*, Paul Farrow a, Christine Favre-Guilmard b, Fraser Hornby a, Sai Man Liu a, Jacquie Maignel-Ludop b, Sandra Marlin a, Imran Mir a, Shilpa Palan a, Mikhail Kalinichev b, Matthew Beard a, Johannes Krupp b. a Ipsen Bioinnovation, Abingdon, Oxfordshire, UK; b Ipsen Innovation, Les Ulis, France * Corresponding author: Ipsen Bioinnovation, 102 Park Drive, Milton Park, Abingdon, OX14 4RY, UK. E-mail address:[email protected].
Introduction: Botulinum neurotoxin serotype B (BoNT/B) is available as a marketed product; however, high doses are required to reach an efficacy similar to that of products containing BoNT/A. Higher doses of BoNT/B have been correlated with an increased incidence of neutralizing antibody production, which is part of the reason for the limited use of the BoNT/B product in the clinic. Recently, a mutation in the light chain of BoNT/B was described that increases the catalytic activity of the isolated BoNT/B light chain in biochemical assays (Guo 2013). In this study, we have produced a full-length recombinant BoNT/B toxin incorporating this mutation and assessed its activity in several biological systems.
Methods: The coding sequence for BoNT/B1 was cloned and expressed in Escherichia coli. The point mutation S201P described by Guo et al was introduced to create the mutated BoNT/B1(S201P). Both mutated and natural sequence toxins were purified and activated by the protease Lys-C. Purified, activated BoNT/B was characterized in cell-free cleavage assays, several cell-based assays, isolated tissue assays, and in the mouse Digit Abduction Score (DAS) test. Results: BoNT/B1(S201P) cleaved VAMP2 in a cell-free assay with higher activity than BoNT/B1, confirming the results of Guo and colleagues. However, there was no significant difference between these two toxins in any of the cell-based assays. Likewise, there was no difference in the inhibition of muscle activity in either of the tissue assays. Finally, in vivo mouse DAS scores as well as in vivo safety parameters were not different between the 2 toxins. Conclusions: Although BoNT/B1(S201P) has a higher catalytic activity than BoNT/B1 in a cell-free system, this does not translate into higher potency in complex biological systems. This probably indicates that the catalytic step is not the rate-limiting step for BoNT/B to reach efficacy. Funding: Ipsen Keywords: BoNT/B; Light chain; Recombinant; VAMP-2 Reference Guo J, Pan X, Zhao Y, Chen S. Engineering clostridia neurotoxins with elevated catalytic activity. Toxicon. 2013;74:158-166. 66. MUSCLE SELECTION FOR ONABOTULINUMTOXINA IN POSTSTROKE LOWER LIMB SPASTICITY INFLUENCES OUTCOME: RESULTS FROM A DOUBLE-BLIND, PLACEBO-CONTROLLED PHASE 3 CLINICAL TRIAL Alberto Esquenazi a, *, Carolyn Geis b, Theodore H. Wein c, Anthony B. Ward d, Chengcheng Liu e, Rozalina Dimitrova f. a MossRehab Gait and Motion Analysis Laboratory, Elkins Park, PA, USA; b Department of Rehabilitation Medicine, Brooks Rehabilitation Physician Group, Daytona Beach, FL, USA; c McGill University, Department of Neurology and Neurosurgery, Montreal General Hospital, Montreal, Quebec, Canada; d North Staffordshire Rehabilitation Centre, Staffordshire University, Stoke on Trent, UK; e Biostatistics, Allergan plc, Bridgewater, NJ, USA; f Neurology and Pain Clinical Development, Allergan plc, Irvine, CA, USA * Corresponding author: MossRehab Gait and Motion Analysis Laboratory, 60 Township Line Road, Elkins Park, PA 19207, USA. E-mail address:aesquena@einstein. edu.
Introduction and Objectives: Injector preference appears to be the main determinant of muscle selection pattern in the treatment of upper-limb spasticity (Baguley 2011). The optimal muscle selection pattern for treatment of poststroke lower limb spasticity (PSLLS) is not yet established. This analysis of the REFLEX study aims to identify an optimal muscle selection pattern for onabotulinumtoxinA (onabotA) injection for the treatment of PSLLS. Methods: The REFLEX study was a phase 3, placebo-controlled study in 60 international centers. Adults with PSLLS (Modified Ashworth Scale [MAS] 3 in the ankle plantar flexors) were eligible for inclusion. During the 12week double-blind phase, patients were randomly assigned to onabotA (300 U into ankle flexors, ie, gastrocnemius, soleus, and tibialis posterior and, optionally, 100 U total into flexor digitorum longus [FDL], flexor digitorum brevis, flexor hallucis longus [FHL], extensor hallucis longus, and/or rectus femoris muscles) or placebo. The primary endpoint, MAS change from baseline, and a secondary endpoint, physician-assessed Clinical Global Impression of Change (CGI), were each reported as the average score of weeks 4 and 6 (the time frame for pharmacologic effect).