Sustained functional benefits after a single set of injections with abobotulinumtoxinA using a 2-mL injection volume in adults with cervical dystonia: 12-week results from a randomized, double-blind, placebo-controlled phase 3b study

Atul T Patel; Mark F Lew; Khashayar Dashtipour; Stuart Isaacson; Robert A Hauser; William Ondo; Pascal Maisonobe; Stefan Wietek; Bruce Rubin; Allison Brashear

doi:10.1371/journal.pone.0245827

. 2021 Feb 1;16(2):e0245827. doi: 10.1371/journal.pone.0245827

Sustained functional benefits after a single set of injections with abobotulinumtoxinA using a 2-mL injection volume in adults with cervical dystonia: 12-week results from a randomized, double-blind, placebo-controlled phase 3b study

Atul T Patel ¹, Mark F Lew ², Khashayar Dashtipour ³, Stuart Isaacson ⁴, Robert A Hauser ⁵, William Ondo ⁶, Pascal Maisonobe ⁷, Stefan Wietek ^8,^*, Bruce Rubin ⁸, Allison Brashear ⁹

Editor: Mandar S Jog¹⁰

¹Kansas City Bone and Joint Clinic, Overland Park, KS, United States of America

²Department of Neurology, Keck/University of Southern California School of Medicine, Los Angeles, CA, United States of America

³Department of Neurology/Movement Disorders, Loma Linda University, Loma Linda, CA, United States of America

⁴Parkinson’s Disease and Movement Disorders Center of Boca Raton, Boca Raton, FL, United States of America

⁵University of South Florida Health Byrd Institute, Parkinson’s Disease and Movement Disorders Center of Excellence, Tampa, FL, United States of America

⁶Methodist Neurological Institute, Houston, TX, United States of America

⁷Ipsen, Boulogne-Billancourt, France

⁸Formerly of Ipsen, Cambridge, MA, United States of America

⁹University of California Davis School of Medicine, Sacramento, CA, United States of America

¹⁰London Health Sciences Centre, CANADA

Competing Interests: I, A. T. Patel, have read the journal’s policy and the authors of this manuscript have the following competing interests: Speaker: Allergan, Ipsen, Merz, Revance; Research support: Allergan, Ipsen, Revance. I, M. F. Lew, have read the journal’s policy and the authors of this manuscript have the following competing interests: Advisor/consultant/speaker: AbbVie, ACADIA, Acorda, Adamas, Cynapsus, Kyowa Kirin, Lundbeck, Neurocrine, Revance, Teva, US WorldMeds; Researcher: Biotie, Enterin Inc., Michael J. Fox Foundation, Parkinson Study Group, Pharm2B; I, A. Brashear, have read the journal’s policy and the authors of this manuscript have the following competing interests: Consulting: Ipsen, Revance; Research support: paid to Wake Forest (institution) and conflicts were managed by Wake Forest. I, K. Dashtipour, have read the journal’s policy and the authors of this manuscript have the following competing interests: Advisor/consultant, and/or speaker: AbbVie, ACADIA, Acorda, Adamas, Allergan, Amneal, Impax, Ipsen, Lundbeck, Neurocrine, Revance, Sunovion, Teva, US WorldMeds. I, S. Isaacson, have read the journal’s policy and the authors of this manuscript have the following competing interests: Honoraria for CME/consultant/promotional speaker: AbbVie, ACADIA, Acorda, Adamas, Addex, Allergan, Amarantus, Axovant, Biogen, Britannia, Eli Lilly, Enterin, GE Healthcare, Global Kinetics, Impax, Intec Pharma, Ipsen, Kyowa, Lundbeck, Michael J. Fox Foundation, Neurocrine, Neuroderm, Parkinson Study Group, Pharma2B, Roche, Sanofi, Sunovion, Teva, UCB, US WorldMeds, Zambon; Honoraria for research grants: AbbVie, ACADIA, Acorda, Adamas, Addex, Allergan, Amarantus, Axovant, Biogen, Britannia, Eli Lilly, Enterin, GE Healthcare, Global Kinetics, Impax, Intec Pharma, Ipsen, Kyowa, Lundbeck, Michael J. Fox Foundation, Neurocrine, Neuroderm, Parkinson Study Group, Pharma2B, Roche, Sanofi, Sunovion, Teva, UCB, US WorldMeds. I, R. A. Hauser, have read the journal’s policy and the authors of this manuscript have the following competing interests: Consulting: AbbVie, Academy for Continued Healthcare Learning, ACADIA, Acorda, Adamas, AstraZeneca, Back Bay Life Science, Biotie, Bracket, Cerecor, ClearView Healthcare Partners, ClinicalMind Medical and Therapeutic Communications, Cowen and Company, Cynapsus Therapeutics, Decision Resources Group, Eli Lilly, eResearch Technology, Expert Connect, Extera Partners, GE Healthcare, Gerson Lehrman Group, Globe Life Sciences, Guidepoint Global, Health Advances, Health and Wellness Partners, HealthLogix, Huron Consulting Group, Impax, Intec Pharma, Jazz Pharmaceuticals, Kyowa Kirin Pharmaceutical Development, LCN Consulting, LifeMax, The Lockwood Group, Lundbeck, MEDACorp, Medscape, Medtronic, Michael J. Fox Foundation, Movement Disorder Society, National Institutes of Health (NIH), Neurocrine Biosciences, Neuroderm, Neuropore Therapies, Outcomes Insights, Parkinson Foundation, Peerview Press, Pennside Partners, Pfizer, Pharma2B, Phase Five Communications, Piper Jaffray & Co, Prexton Therapeutics, Projects in Knowledge, Putnam Associates, Quintiles, RMEI Medical Education for Better Outcomes, Sarepta Therapeutics, Schlesinger Associates, Scion Neurostim, Seagrove Partners, Slingshot Insights, Sun Pharma, Sunovion, Teva, US WorldMeds, Vista Research, WebMD, Windrose Consulting Group; Research support: AbbVie, Acorda Therapeutics, AstraZeneca, Axovant Sciences, Biogen, Cavion, Dart NeuroScience, Enterin, F. Hoffman-La Roche, Impax, Intec Pharma, Jazz Pharmaceuticals, Lundbeck, Michael J. Fox Foundation, NeuroDerm, Parkinson’s Foundation, Prexton Therapeutics, Revance, Sunovion. I, W. Ondo, have read the journal’s policy and the authors of this manuscript have the following competing interests: Advisor/consultant/speaker: ACADIA, Acorda, Adamas, Jazz Pharmaceuticals, Neurocrine, Teva, UCB, US WorldMeds; Research support: Biogen, Lilly, Lundbeck, Sun Pharmaceuticals, Sunovian. I, P. Maisonobe, have read the journal’s policy and the authors of this manuscript have the following competing interests: Employment: Ipsen. I, S. Wietek, have read the journal’s policy and the authors of this manuscript have the following competing interests: Employment: Ipsen. I, B. Rubin, have read the journal’s policy and the authors of this manuscript have the following competing interests: Employment (former): Ipsen.

^✉

* E-mail: Stefanwietek15@gmail.com

Roles

Atul T Patel: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

Mark F Lew: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

Khashayar Dashtipour: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

Stuart Isaacson: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

Robert A Hauser: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

William Ondo: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

Pascal Maisonobe: Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing

Stefan Wietek: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

Bruce Rubin: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

Allison Brashear: Conceptualization, Investigation, Methodology, Writing – original draft, Writing – review & editing

Mandar S Jog: Editor

PMCID: PMC7850472 PMID: 33524060

Abstract

Cervical dystonia (CD) is primarily treated with botulinum toxin, at intervals of ≥ 12 weeks. We present efficacy, patient-reported outcomes (PROs), and safety in adults with CD at the last available visit after a single set of abobotulinumtoxinA (aboBoNT-A) injections versus placebo using 500 U in a 2-mL injection volume. In this 12-week, randomized, double-blind trial, patients were ≥ 18 years of age with primary idiopathic CD, had a Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) total score ≥ 20, and TWSTRS-Severity subscale score > 10 at baseline. Patients (N = 134) were randomized (2:1) to aboBoNT-A (n = 89) or placebo (n = 45), with aboBoNT-A patients treated with 500 units (U) if toxin-naïve, and 250 to 500 U based on previous onabotulinumtoxinA dose if non-naïve. Endpoints included total TWSTRS, Pain Numeric Rating Scale (NRS-Pain; 24-hour), Treatment Satisfaction Questionnaire for Medication, and other PROs for pain, depression, and global health. Results are for the intent-to-treat population, with “Week 12” (Wk12) comprising the last available post-baseline assessment (end-of-study or early withdrawal). Mean TWSTRS total scores improved from 42.5 at baseline to 35.4 at Wk12 with aboBoNT-A and 42.4 to 40.4 with placebo (treatment difference: –4.8; 95% confidence interval [CI]: –8.5, –1.1; p = 0.011). At Wk12, mean (95% CI) change from baseline in NRS-Pain was –1.0 (–1.59, –0.45) for aboBoNT-A and –0.2 (–0.96, 0.65) for placebo. AboBoNT-A demonstrated numeric improvements in other PROs. More aboBoNT-A–treated patients than patients receiving placebo reported being at least “somewhat satisfied” with treatment (60.4% vs 42.2%, respectively), symptom relief (57.0% vs 40.0%), and time for treatment to work (55.8% vs 33.3%). No new adverse events were reported. Results indicate that in patients with CD, treatment with aboBoNT-A using a 2-mL injection provided sustained improvement in the TWSTRS total score and patient-perceived benefits up to 12 weeks.

Trial registration: Clinicaltrials.gov Identified: NCT01753310.

Introduction

Cervical dystonia (CD), a chronic neurologic movement disorder characterized by sustained or repetitive involuntary contractions of the neck muscles that leads to abnormal postures, represents the most common focal dystonia. The prevalence of CD worldwide has been estimated to range from 20 to 4,100 cases per million [1]. Intramuscular injection of botulinum neurotoxin into affected muscles represents the primary treatment for CD [2]. Botulinum neurotoxin type A binds to receptors on peripheral cholinergic nerve endings and is internalized by receptor-mediated endocytosis. This is followed by cleavage of SNAP25, a protein needed for vesicle fusion to the presynaptic membrane, which is required for synaptosomal release of acetylcholine into the neuromuscular junction. The efficacy and safety of abobotulinumtoxinA (aboBoNT-A) for the treatment of CD has been reported based on two randomized, controlled trials and their open-label safety extensions [3–5]. AboBoNT-A is approved in the United States of America (US) and Europe for the treatment of adults with CD [6, 7].

Initially, the prescribing information in the US specified a 500-unit (U), 1-mL dilution regimen for AboBoNT-A administration; however, feedback obtained from scientific experts, community injectors, and market research studies favored use of a 500 U, 2-mL dilution. This was most likely related to prior familiarity in using comparable volumes with other approved toxins in the US.

The 2-mL dilution regimen has since been approved in the US based on the results of a 12-week, phase 3b, randomized, double-blind, placebo-controlled clinical trial that was conducted to evaluate the efficacy and safety of a 500 U, 2-mL dilution of aboBoNT-A versus placebo in adults with CD. AboBoNT-A improved the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) total score at Week 4. AboBoNT-A demonstrated significant improvements in symptoms in both toxin-naïve and previously treated patients; a safety profile similar to the 1-mL dilution was observed [8]. An open-label extension study (NCT01753336) provided long-term safety and efficacy data, which also supported the US approval of the 2-mL dilution regimen. This manuscript expands on the 4-week results of the previously published placebo-controlled trial by providing exploratory data for up to 12 weeks to assess the durability of response with aboBoNT-A 500 U using a 2-mL dilution. This study was registered on ClinicalTrials.gov (NCT01753310).

Methods

Subjects were given a full explanation, in lay terms, of the aims of the study, the benefits, potential discomforts and risks of taking part in the study prior to enrollment. A written explanation was also provided and written informed consent was obtained prior to enrollment. The study protocol, subject information leaflet, and informed consent document were reviewed and approved by Harrison IRB (London, OH) as well as by institutional review boards at individual study sites (if locally required) prior to commencement of the study.”

This study was conducted under the provisions of the Declaration of Helsinki, informed consent regulations and in accordance with the International Conference on Harmonisation (ICH) Consolidated Guideline on Good Clinical Practice (GCP) and local site review boards. This study was registered with ClinicalTrials.gov (identifier: NCT01753310).

Study design

This was a 12-week, phase 3b, multicenter, randomized, double-blind, placebo-controlled study, conducted at 43 initiated sites in the US, 38 of which enrolled patients (S1 Fig). Patients were randomized (2:1) to receive aboBoNT-A or placebo by intramuscular injection, stratified by whether the patient had previous treatment with onabotulinumtoxinA (onaBoNT-A). Toxin-naïve patients who were randomized to the aboBoNT-A group received 500 U, using the 2-mL dilution method in at least two affected neck muscles; patients with prior onaBoNT-A exposure (non-naïve at baseline) received 250 to 500 U, using the 2-mL dilution method (based on previous onaBoNT-A dose) into previously injected muscles.

Patients

Eligible patients included adults (aged ≥ 18 years) with a primary diagnosis of idiopathic CD (duration ≥ 9 months) and with a TWSTRS total score ≥ 20 and a TWSTRS severity subscale score > 10 at baseline. Patients could be BoNT-A naïve or could have received any other formulation of BoNT-A, including aboBoNT-A, prior to study enrollment as long as their last two injections were onaBoNT-A and they had a satisfactory clinical response to both injections.

Treatment

All patients received a single set of intramuscular injections of either aboBoNT-A (500 U/2 mL) or placebo in a minimum of two clinically affected neck muscles. The sites and the dose per site were determined by the investigator according to the standard practice and disease presentation. Electromyography-guided injections were allowed at the preference of the investigator at each site. Patients randomized to the aboBoNT-A group received 500 U of aboBoNT-A if they were onaBoNT-A treatment-naïve or 250–500 U of aboBoNT-A at a 2.5:1 (aboBoNT-A:onaBoNT-A) ratio to their previous onaBoNT-A dose into muscles injected during the last two sequential cycles of onaBoNT-A within the past 18 months for the treatment of CD. The amount of aboBoNT-A injected into the sternocleidomastoid (SCM) muscle was limited to ≤ 0.6 mL (150 U), in order to reduce the occurrence of dysphagia, per the aboBoNT-A US prescribing information [6]. Prior to administration, aboBoNT-A and placebo vials were reconstituted at the investigational site with 2-mL preservative-free sodium chloride for injection (0.9%). Detailed instructions were provided for the volume that needed to be withdrawn from the reconstituted aboBoNT-A vials.

Assessments

The primary efficacy endpoint was change from baseline in TWSTRS total score at Week 4, with secondary endpoints in the testing hierarchy comprising TWSTRS total score, TWSTRS response, Patient Global Impression of Change (PGIC), and the Cervical Dystonia Impact Profile-58 (CDIP-58) at Week 2 and Week 4. Tertiary efficacy endpoints included assessments of the change from baseline at Week 12.

The TWSTRS total score comprises three subscale scores: severity, disability, and pain, each of which is scored independently and can have a value from 0 to 85 (best to worst) [9]. The TWSTRS total score was used to assess the severity of CD and was assessed by the investigator prior to study treatment at baseline (Day 1) and at all post-treatment visits. PGIC was assessed using a 7-point Likert scale, ranging from +3 (very much improved) to –3 (very much worse). The Pain Numeric Rating Scale (NRS-Pain) is a numeric, 24-hour assessment scale that measures pain as 0 (no pain) to 10 (worst possible pain). The Brief Pain Inventory (BPI) short form was used to assess the effect of CD pain on seven areas (general activity, mood, walking ability, normal work, relations with other people, sleep, and enjoyment of life) and ranges from 0 (does not interfere) to 10 (completely interferes).

Sample size

It was determined that 132 randomized patients would be sufficient to demonstrate the superiority of aboBoNT-A to placebo assuming a minimum clinically relevant difference in the adjusted least squares mean change from baseline in TWSTRS total score at Week 4 between aboBoNT-A 2-mL and placebo equal to 5.5, a common standard deviation (SD) in the change from baseline in TWSTRS total score at Week 4 equal to 8.8, a power of 90%, a two-tailed type I error equal to 5%, and 10% dropout rate.

Randomization

The sponsor’s randomization manager, who was a statistician independent from the study, prepared two lists that were performed in blocks and were based on a computer-generated randomization list. The first list of randomization numbers stratified for subjects who were BoNT-A treatment naïve or non-naïve at baseline was generated with a 2:1 ratio of aboBoNT-A:onaBoNT-A. The second was a list of treatment numbers, which were specified on the treatment packs, to be dispatched to the sites in order to dispense the drug. This list was produced on a 1:1 basis (aboBoNT-A:onaBoNT-A). The randomization, as well as the treatment number assignation at drug dispensation, was managed by an Interactive Web Response System (IWRS) service. At screening, potential patients were assigned a patient number. Following confirmation of eligibility, patients were given a randomization/treatment allocation number and were assigned to one of the two groups (aboBoNT-A or placebo) at baseline, in sequential order within each site (and within each level of strata). This was a single-dose study. The sites of injection and the dose per site were determined by the investigator according to the standard practice and disease presentation.

Blinding

Patients and investigators were kept blinded to the allocation. Study treatments were similar in size, color, smell, and appearance, allowing the blinded conditions of the study to be maintained. A set of individually sealed code-break envelopes was prepared by the sponsor’s randomization manager to enable emergency code-break procedures of individual patients without compromising the blinding of the study and was provided to the Central Department of Pharmacovigilance at Ipsen.

Statistical analysis

The statistical analysis of the efficacy and safety data was performed using Statistical Analysis Software (SAS; version 9.2). The intent-to-treat (ITT) population included all randomized patients; the modified ITT population included randomized patients with both a baseline and Week 4 post-treatment TWSTRS total score assessment. Efficacy results are presented for the ITT population (all randomized patients). The last available post-baseline assessment was either the end of study (Week 12) or early withdrawal. Withdrawals included patients who exited the study early and patients who began the open-label protocol due to lack of efficacy after 4 weeks.

For TWSTRS total and subscores, the change from baseline is expressed as weighted overall treatment difference and 95% confidence interval (CI). Significance testing for the superiority of aboBoNT-A versus placebo was conducted for the primary and secondary efficacy endpoints at a two-tailed significance level of 5% by using a stratified analysis of covariance (ANCOVA) with baseline TWSTRS total score as covariate and stratified by the randomization stratification factor. The hierarchical testing procedure was stopped if there was no statistically significant treatment effect on the primary efficacy endpoint. Otherwise, the superiority of aboBoNT-A was then tested for each secondary endpoint in rank order, proceeding to the next endpoint only when treatment effect on the current endpoint was deemed statistically significant. Statistics were summarized by treatment group for tertiary endpoints but were not compared by formal statistical testing.

Data sharing

Where patient data can be anonymized, Ipsen will share all individual participant data that underlie the results reported in this article with qualified researchers who provide a valid research question. Study documents, such as the study protocol and clinical study report, are not always available. Proposals should be submitted to DataSharing@Ipsen.com and will be assessed by a scientific review board. Data are available beginning 6 months and ending 5 years after publication; after this time, only raw data may be available.

Results

Patient disposition and study exposure

A total of 134 patients were randomized and included in the ITT population (aboBoNT-A, n = 89; placebo, n = 45) (Table 1). One patient was excluded from the analysis because he did not receive study drug. From the aboBoNT-A group 87 patients (98%) attended Week 2 visits, 84 (94%) attended Week 4, and 57 (64%) attended Week 12. In the placebo group 45 patients (100%) attended Week 2 visits, 45 (100%) attended Week 4, and 21 (47%) attended Week 12 (Fig 1). Forty-seven patients (aboBoNT-A, n = 25 [28%]; placebo, n = 22 [49%]) elected to enter the open-label portion of the study prior to Week 12, with 33 entering the open-label study at Week 4. Table 2shows a post-hoc analysis of patient study exposure based on all ITT patients and all of those who withdrew from the study early.

Table 1. Baseline demographics and characteristics (ITT populations).

	aboBoNT-A	Placebo
	n = 89	n = 45
Female sex, n (%)	59 (66.3)	28 (62.2)
Age (years; mean ± SD)	57.3 ± 11.11	56.5 ± 11.74
Caucasian/white race, n (%)	84 (94.4)	42 (93.3)
Type of CD, n (%)
Torticollis	75 (84.3)	39 (86.7)
Laterocollis	54 (60.7)	30 (66.7)
Anterocollis	17 (19.1)	7 (15.6)
Retrocollis	20 (22.5)	8 (17.8)
Lateral shift	22 (24.7)	14 (31.1)
Sagittal shift	9 (10.1)	8 (17.8)
Previous exposure to botulinum neurotoxin, n (%)
Yes	57 (64.0)	29 (64.4)
No	32 (36.0)	16 (35.6)
Previous botulinum neurotoxin treatments for CD, n (%)
n^*	57/57 (100.00)	29/29 (100.0)
onaBoNT-A	56/57 (98.2)	28/29 (96.6)
aboBoNT-A	2/57 (3.5)	1/29 (3.4)
incoBoNT-A	6/57 (10.5)	2/29 (6.9)
rimaBoNT-B	5/57 (8.8)	2/29 (6.9)
Most recent CD treatment with onaBoNT-A^**
n	55	25
Mean dose^*** (U; mean ± SD)	177.0 ± 34.0	176.2 ± 42.2
Mean time since last injection (months; mean ± SD)	4.8 ± 19.1	3.6 ± 16.3

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*Subjects may have been previously treated with ≥1 botulinum neurotoxin.

**In subjects who were non-naïve for onaBoNT-A treatment.

***Mean dose of onaBoNT-A exceeds 200 U eligibility requirement due to protocol deviations, patients were excluded from ITT.

aboBoNT-A = abobotulinumtoxinA; CD = cervical dystonia; incoBoNT-A = incobotulinumtoxinA; ITT = intent-to-treat; onaBoNT-A = onabotulinumtoxinA; rimaBoNt-B = rimabotulinumtoxinB; SD = standard deviation.

Fig 1 — aboBoNT-A = abobotulinumtoxinA; HCP = healthcare provider; mITT = modified intent-to-treat; OLE = open-label extension; PP = per protocol; TWSTRS = Toronto Western Spasmodic Torticollis Rating Scale. *Entered NCT01753336 (OLE) to continue active treatment.

Table 2. Patient (ITT) study exposure for all patients and patients who withdrew from the study.

ITT Population
Parameter	AboBoNT-A	Placebo	Total
N	89	45	134
Mean (SD) days	67.7 (38.4)	52.5 (34.7)	62.6 (37.8)
CI 95%	59.6; 75.8	42.1; 62.9	56.2; 69.1
Median days	85.0	35.0	85.0
Min; Max	1.0; 184.0	14.0; 105.0	1.0; 184.0
ITT Patients Who Withdrew From Study
N	32	24	56
Mean (SD) days	19.8 (8.4)	22.0 (8.6)	20.7 (8.5)
CI 95%	16.7; 22.8	18.4; 25.6	18.4; 23.0
Median days	15.5	18.0	16.0
Min; Max	1.0; 37.0	14.0; 45.0	1.0; 45.0

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aboBoNT-A = abobotulinumtoxinA; CI = confidence interval; ITT = intent-to-treat; SD = standard deviation.

Overall study duration took place from January 2013 to January 2015, which included patient recruitment (approximately 9 months) and the last patient follow-up (up to 4 months). Individual treatment duration was between 4 weeks and 16 weeks, due to the study design, which allowed a ±2 day window to complete the Week 2 and 4 visits, and a 28 day window to complete the Week 12 visit.

TWSTRS

Previously reported mean (SD) TWSTRS total score at baseline was 42.5 (10.40) for aboBoNT-A and 42.4 (10.63) for placebo; significant decreases in TWSTRS total score with aboBoNT-A versus placebo were observed at Week 2, with further improvements at Week 4 [8]. For the last available data at Week 12 or at early study withdrawal, the weighted overall treatment difference between aboBoNT-A and placebo was also statistically significant (–4.8; 95% CI: –8.5, –1.1; p = 0.011; Fig 2). As tertiary endpoints, the TWSTRS subscales were not compared by formal statistical testing; however, at all of the time points, aboBoNT-A treatment demonstrated numerically greater reductions in the TWSTRS-Severity, TWSTRS-Pain, and TWSTRS-Disability subscale scores.

Fig 2 — aboBoNT-A = abobotulinumtoxinA; ITT = intent-to-treat (all randomized patients); last available* = last available post-baseline (end of study or early withdrawal); TWSTRS = Toronto Western Spasmodic Torticollis Rating Scale; Δ = weighted overall treatment difference. Error lines indicate standard deviation. *Last available = last available post-baseline (end of study or early withdrawal), mean (SD) study drug exposure: 62.6 (37.8) days.

Patient-reported outcomes

With the exception of CDIP-58 at Week 2 and Week 4, patient-reported outcomes (PROs) were tertiary endpoints and were not compared by formal statistical testing. At all of the time points, the majority of aboBoNT-A–treated patients indicated minimal improvements or greater via PGIC scores, at a rate approximately twice that of placebo (Fig 3A). More patients receiving placebo than aboBoNT-A–treated patients indicated no change or worse than at baseline. At Week 4, 34.1% and 31.8% of patients receiving placebo reported no change or worsening, respectively. For a more detailed analysis of PGIC at each time point, see S3 Fig. In the patient-reported rating of pain reflecting the previous 24 hours, the mean change from baseline in NRS-Pain was Δ –1.0 (95% CI: –1.59, –0.45) for aboBoNT-A and Δ –0.2 (95% CI: –0.96, 0.65) for placebo, with a consistent reduction from baseline at Week 2 and Week 4 for aboBoNT-A–treated patients, indicating reduced pain (Fig 3B). AboBoNT-A also demonstrated numeric benefits on the BPI assessment (Fig 3C), although the trajectory was similar in both treatment groups.

Fig 3 — Patient-reported outcomes (ITT population): A) PGIC across time points. B) Mean NRS-Pain scores across time points. C) Mean BPI scores across time points. D) Level of depressive (PHQ-9) mood across time points. E) Change from baseline in CDIP-58 scaled sum total score across time points. BPI = Brief Pain Inventory; CDIP-58 = Cervical Dystonia Impact Profile-58; ITT = intent-to-treat (all randomized patients); Improved = minimally, much, or very much improved; Last available* = last available post-baseline (end of study or early withdrawal); Mild/moderate = mild depression (5‒9) or moderate depression (10‒14); No/minimal = no depression (0) or minimal depression (1‒4); NRS-Pain = numeric rating scale for pain; Worse = minimally, much, or very much worse; PGIC = Patient Global Impression of Change; PHQ-9 = Patient Health Questionnaire-9; Severe = moderately severe depression (15‒19) or severe depression (20–27). * Last available = last available post-baseline (end of study or early withdrawal), mean (SD) study drug exposure: 62.6 (37.8) days.

For aboBoNT-A–treated patients, there was a consistent shift toward less depression from baseline to Week 2 and continuing through Week 4 and the last available assessment (Fig 3D). At Week 2, 59.4% of aboBoNT-A–treated patients had no or minimal depression compared with 47.3% of patients receiving placebo. At Week 4, the rates were 63.0% and 48.6% for aboBoNT-A and placebo, respectively. At the last available assessment, 61.0% of aboBoNT-A–treated patients had no or minimal depression compared with 46.0% of patients receiving placebo. The change from baseline in the CDIP-58–scaled sum total score was consistently greater with aboBoNT-A than with placebo (Fig 3E). At Week 2 aboBoNT-A was Δ –5.8 (95% CI: –8.85, –2.76) compared with placebo at Δ –4.3 (95% CI: –8.79, 0.13). At Week 4 aboBoNT-A was Δ –8.5 (95% CI: –11.61, –5.30) compared with placebo at Δ –4.7 (95% CI: –9.60, 0.24) (p = not significant). At the last available assessment, aboBoNT-A was Δ –7.9 (95% CI: –11.01, –4.70) compared with placebo at Δ –3.1 (95% CI: –7.75, 1.45) (p = not significant).

Satisfaction

Across time points, approximately 65% of aboBoNT-A–treated patients were at least somewhat satisfied or greater with the medication overall according to results of the modified Treatment Satisfaction Questionnaire for Medication (TSQM-9). Patients receiving placebo reported lower satisfaction rates, with 32.5% at Week 2, 35.6% at Week 4, and 40.0% of patients at the last available assessment being at least somewhat satisfied or more. Regarding satisfaction with symptom relief, 54.1% of aboBoNT-A–treated patients were at least somewhat or more satisfied at Week 2, increasing to 58.1% at Week 4 and 57.0% for last available assessment. Rates for placebo decreased from Week 2 (28.0%) to Week 4 (24.4%), with 40.0% of patients receiving placebo being at least somewhat satisfied with symptom relief at the last available assessment. More than half of aboBoNT-A–treated patients were at least somewhat satisfied with the length of time it took for medication to work (Week 2, 53.0%; Week 4, 59.3%; last available, 55.8%). Only 23.3% of patients receiving placebo were at least somewhat satisfied at Week 2, decreasing to 20.0% at Week 4, and increasing to 33.3% for the last available assessment.

Safety

The treatment-emergent adverse events (TEAEs) are reported in Table 3[7]. For those receiving aboBoNT-A, 41% of patients reported TEAEs, compared with 22% of patients receiving placebo. In the aboBoNT-A group, one patient discontinued the study due to the emergence of a TEAE (colon neoplasm) not thought to be related to study medication. No patients in the placebo group discontinued the study due to an AE [8].

Table 3. Treatment-emergent adverse events in the safety set.

Event, n (%)	aboBoNT-A	Placebo
Event, n (%)	n = 88 (100.0)	n = 45 (100.0)
All treatment-emergent adverse events	36 (40.9)	10 (22.2)
Dysphagia	8 (9.1)	0 (0.0)
Muscular weakness	8 (9.1)	0 (0.0)
Neck Pain	7 (8.0)	0 (0.0)
Headache	5 (5.7)	0 (0.0)
Sinusitis	3 (3.4)	0 (0.0)
Bronchitis	2 (2.3)	0 (0.0)
Burning sensation	2 (2.3)	0 (0.0)
Depression	2 (2.3)	1 (2.2)
Diarrhea	2 (2.3)	0 (0.0)
Fatigue	2 (2.3)	0 (0.0)
Blurred vision	2 (2.3)	0 (0.0)
Serious Adverse Events	4 (4.5)	1 (2.2)
Dysphagia	1 (1.1)	0 (0.0)
Colon neoplasm	1 (1.1)	0 (0.0)
Endometrial cancer	1 (1.1)	0 (0.0)
Transient ischemic attack	1 (1.1)	0 (0.0)
Depression	0 (0.0)	1 (2.2)

Open in a new tab

aboBoNT-A = abobotulinumtoxinA.

Discussion/conclusion

These results demonstrate the sustained benefit of treatment with aboBoNT-A 500 U/2-mL dilution for up to 12 weeks in patients with CD, further supporting the use of the 2-mL dilution regimen and allowing dilution flexibility for clinicians.

In order to better understand how early withdrawal may have impacted the last available visit analysis, a descriptive analysis of TWSTRS at Week 12 was performed based only on patients who completed the study and those who withdrew early (with available TWSTRS data at week 12). The unadjusted difference between placebo and aboBoNT-A is 4.7 in favor of aboBoNT-A. These results are very similar to the results from the last available assessment analysis (4.8), demonstrating the benefit of aboBoNT-A at Week 12.

Efficacy of the 2-mL dilution of aboBoNT-A is similar to results from previous studies of 1-mL dilution of aboBoNT-A 500 U [3, 4]. For both of the trials using the 1-mL dilution, aboBoNT-A was significantly more effective than placebo in the change from baseline in TWSTRS score. The adjusted mean difference in the current study was −8.3 at Week 4, which is similar to the −6.0 and −8.8 observed in the pivotal clinical trial at Week 4. The weighted overall treatment difference between aboBoNT-A and placebo was –4.8 at Week 12 or at early study withdrawal in the current trial, similar to the adjusted mean difference of –4.3 and -4.1 in the pivotal clinical trials at Week 12.

The safety profile of the 500 U/2-mL aboBoNT-A dilution is similar to that observed with the 500 U/1-mL dose used in the aboBoNT-A pivotal trial. In the current study, TEAEs were reported in 41% of patients receiving aboBoNT-A, with the most common being dysphagia (9.1%), muscle weakness (9.1%), neck pain (8.0%), and headache (5.7%); in the aboBoNT-A 500 U/1-mL pivotal trial the most common AEs were dysphagia (9%), neck pain (5%), injection site pain (5%), headache (4%), and upper respiratory tract infection (4%) [4].

Limitations of the study include the use of a fixed dose of aboBoNT-A, which is an important aspect and was done to demonstrate how BoNT-A naïve patients would respond to a standard starting dose. This dosing method differs from that used in clinical practice, where doses are individualized for each patient, and it is likely that many of the participants did not receive the optimal dose in the appropriate muscle. Nonetheless, the use of a fixed dose was necessary, as there were no data available on which to base dose optimization in naïve patients. Additional limitations of this study include the study design allowing patients who were deemed eligible to enter into the open-label extension study between Week 4 and Week 8 (before they reached the planned Week 12 end of the study visit), which resulted in 25 patients withdrawing to enter the open-label extension.

The data reported here indicate that treatment with aboBoNT-A 500 U/2-mL dilution provided patient-perceived benefits through 12 weeks. AboBoNT-A–treated patients experienced decreased pain, decreased depression, and improved health—factors associated with increased quality of life in CD patients. Furthermore, PROs were maintained over time, in line with the improved disease state. These positive outcomes support the administration of aboBoNT-A 500 U using a 2-mL dilution.

Supporting information

S1 Fig. Study site locations.

(DOCX)

Click here for additional data file.^{(16.8KB, docx)}

S2 Fig

Mean TWSTRS sub-scale scores across time points (ITT population): A) Mean TWSTRS severity score across time points. B) Mean TWSTRS pain score across time points. C) Mean TWSTRS disability score across time points. Tertiary endpoints: no formal statistical testing was conducted. ITT = intent-to-treat (all randomized patients); last available = last available post-baseline* (end of study or early withdrawal); TWSTRS = Toronto Western Spasmodic Torticollis Rating Scale. Error bars represent the 95% confidence interval. *Last available = last available post-baseline (end of study or early withdrawal), mean (SD) study drug exposure: 62.6 (37.8) days.

(PNG)

Click here for additional data file.^{(34.5KB, png)}

S3 Fig

Patient Global Impression of Change at (A) Week 2, (B) Week 4, and (C) last available (ITT). A. Week 2. B. Week 4. C. Last available. *Last available = last available post-baseline (end of study or early withdrawal), mean (SD) study drug exposure: 62.6 (37.8) days.

(PNG)

Click here for additional data file.^{(29.5KB, png)}

S1 File

(PDF)

Click here for additional data file.^{(818.7KB, pdf)}

Acknowledgments

The authors thank all patients involved in the study, as well as investigators and research staff in participating institutions, and Jim Otto, for his substantial contributions to the study while he was employed at Ipsen. The authors also thank Nicole Coolbaugh, BSc, CMPP, Kate Katsaval, BSc, and Philip Sjostedt, BPharm, of The Medicine Group, LLC (New Hope, PA, USA) for providing medical writing support.

Data Availability

The data underlying the study's results cannot be shared publicly due to participant confidentiality. However, other researchers who provide a valid research question may request access to the data by submitting a proposal to DataSharing@Ipsen.com. Proposals will be assessed by a scientific review board.

Funding Statement

Ipsen, Cambridge, MA, USA provided support for this study in the form of funding for medical writing support in accordance with current Good Publication Practice guidelines (GPP3) and salaries for PM, SW, and BR. The specific roles of these authors are articulated in the ‘author contributions’ section. The funder had no additional role in the study design, data collection and analysis, or decision to publish.

References

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7.EMA List of Approved Medicinal Products. 2019. Active substance: botulinum toxin a—haemagglutinin complex. Date accessed: 11/4/2020. Available from: https://www.ema.europa.eu/en/documents/psusa/botulinum-toxin-haemagglutinin-complex-list-nationally-authorised-medicinal-products-psusa/00000427/201812_en.pdf.
8.Lew MF, Brashear A, Dashtipour K, Isaacson S, Hauser RA, Maisonobe P, et al. A 500 U/2 mL dilution of abobotulinumtoxinA vs. placebo: randomized study in cervical dystonia. Int J Neurosci. 2018;128(7):619–26. 10.1080/00207454.2017.1406935 [DOI] [PubMed] [Google Scholar]
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PLoS One. doi: 10.1371/journal.pone.0245827.r001

Decision Letter 0

Nancy Beam

28 Sep 2020

PONE-D-19-35636

Sustained functional benefits after a single set of injections with abobotulinumtoxinA using a 2-mL injection volume in adults with cervical dystonia: 12-week results from a randomized, double-blind, placebo-controlled phase 3b study

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Reviewer #1: In this study, the authors present findings on the use of abobotulinum toxin for cervical dystonia when 2ml dilution is used for preparation. The authors present data for outcomes at 12 weeks. They have already published the outcomes at four weeks follow-up. In their analysis of primary outcomes, the authors found that active group continued to have significant benefits at 12 weeks follow-up compared to the placebo. These findings indicate there are longer than expected benefits as most effects related to botulinum are expected wane at 12 weeks follow-up.

The discussion section is quite underdeveloped. Are these 12 week-outcomes different from those seen with 1ml dilution when evaluated at a similar interval of follow-up? The authors draw comparisons between 1 ml and 2 ml dilution for four weeks' follow-up. The authors should provide scientific insights to understand the value of their findings.

Reviewer #2: Last line of abstract: if most patients were measured at 12 weeks, this would be better as “at 12 weeks” rather than “up to 12 weeks”.

Pg 14- “treatment duration was between 4 and 16 weeks” - wasn’t it a single injection?

Reviewer #3: This study compares efficacy, patient-reported outcomes, and safety in CD patients receiving 2-ml aboBoNT-A or 2-ml placebo. It is shown that a treatment with 2-ml aboBoNT-A is superior compared to placebo. Besides, the results of the actual study are compared with the results of a previous 1-ml aboBoNT-A study. Since the results are comparable, the administration of aboBoNT-A 500 U using a 2-ml dilution is recommended.

My main criticism of the study is that the design and the limitations of this study have to be discussed.

Comments in detail:

Abstract:

It is only the dilution (2 ml) mentioned but not the diluted units. Please add so that the concentration is clear.

Introduction:

(page 7, line 6): ‘AbobotulinumtoxinA (aboBoNT-A; Dysport®) inhibits acetylcholine release and binds to receptors on peripheral cholinergic nerve endings, and is internalized by receptor-mediated endocytosis. This is followed by cleavage of SNAP25, a protein needed for vesicle fusion to the presynaptic membrane, which is required for synaptosomal release of neurotransmitter into the neuromuscular junction.’

This part describes the mode of action of botulinum neurotoxin in general and not of aboBoNT-A in special. So, I would recommend to change in line 6 from ‘AbobotulinumtoxinA’ to botulinum neurotoxin. Besides, the first sentence starts with the final result of the effect of BoNT-A and then goes further to the single steps of the mode of action. I would recommend to delete the first part of the sentence and to start with ‘Botulinum neurotoxin binds to…’ and change in the end of the following sentence from ‘neurotransmitter’ to ‘acetylcholine’.

Last sentence of first paragraph: AboBoNT-A is not only approved in the USA but also in Europe for cervical dystonia. This should be also mentioned.

Second paragraph: This part is a little confusing because only the volume but not the diluted BoNT-A dosis is mentioned. So, the concentration remains unclear. Since there are different vials (300 U and 500 U available) the U per ml has to be mentioned.

Methods

Treatment (line 7): oba should be changed to ona

Treatment: Regarding your study protocol the onaBoNT-A dosis was converted to aboBoNT-A dosis using a factor of 2.5. This means that the maximal dosis of onaBoNT-A was 200 U since the maximal dosis of aboBoNT-A in your study is limited to 500 U. I would like to know what happened with patients that received more than 200 U of onaBoNT-A (in table 1 it is stated that mean dose of onaBoNT-A was 177+/-34 respectively 176 +/- 42 U. So, in both treatment groups there were onaBoNT-A treated patients that received more than 200 U onaBoNT/A). Was the calculated aboBoNT-A dosis (factor 2.5:1) reduced or have these patients more than 500 U aboBoNT-A received? Please state in the Methods what you have done in such a case.

On the other hand, all BoNT-A native patients received 500 U aboBoNT-A. It is stated that ‘the sites and the dose per site were determined by the investigator according to the standard practice and disease presentation.’ But the total doses were given by the study protocol. I find this difficult because not every patient needs 500 U (corresponding to your patients that were treated with onaBoNT-A before). More explanations to the clinical usage of the fix total dose regiment in your study would be helpful.

Results

Figures: there are too many pixels. It is not possible to zoom in and to read the figures easily. Especially in figure 3B: the headings are to bold. It is not possible to read them.

Please improve.

Discussion

Third paragraph: ‘TWSTERS’ should be changed to ‘TWSTRS’

I think it is very important that a paragraph with the limitations of the study is added.

It should be discussed why only BoNT-A native patients or onaBoNT-A treated patients were included in the study. Why did you not include 1-ml aboBoNT-A treated patients and changed to a 2-ml aboBoNT-A treatment? Besides, the direct comparison between 1-ml and 2-ml aboBoNT-A treatment would be interesting. This would be more interesting than the comparison of effects of BoNT-A versus placebo. But you have compared the results of two different studies (1-ml versus 2-ml compared to placebo, respectively). So, it is not surprising that two different studies show comparable effects. It would be interesting to investigate this in one study. The result of your study that BoNT-A is the better treatment compared to placebo is not new. Please discuss this methodological difficulty of your paper.

Altogether it is not very surprising that CD patients improve when they are injected with BoNT-A compared to placebo. Patients with BoNT-A receive the ‘primary treatment for CD’ as you have stated in your introduction. It is known from literature that there are only little to no differences when the volume of BoNT-A is only doubled. To reach a volume effect the volume has to be increased much higher. Please discuss with the current literature.

Reviewer #4: This is a randomised, double-blind, placebo-controlled trial on sustained functional benefits after a single set of injections with abobotulinumtoxinA using a 2-mL injection volume in adults with cervical dystonia.

The aim of the authors was to assess the efficacy, patient-reported outcomes (PROs), and safety in adults with CD after a single set of abobotulinumtoxinA (aboBoNT-A) injections versus placebo after a single set of injections using 2-mL injection volume. 134 patients were randomised and received aboBoNT-A 500 units (U) if toxin-naïve, and 250 to 500 U based on previous onabotulinumtoxinA dose if non-naïve patients.

The conclusions were that there was a sustained improvement in TWSTRS score and patient-perceived benefits up to 12 weeks with abobotulinumtoxinA group. There was also improvement in treatment satisfaction, pain, depression, and global health.

In general, this is a complete and well written about sustained abobotulinumtoxinA efficacy in cervical dystonia.

Reviewer #5: Overall impression: I found this paper unclear to read. It is also too long for a very simple conclusion that a 2ml dilution also works. I am not convinced that this small conclusion requires a rehash of the entire study that has already been published. In my mind this is a short report only. Much of the actual 12week data is presented subjectively with very mixed if any statistics, lots of drops outs, and early treatments etc and is truly hard to interpret. I would strongly suggest the authors to substantially shorten the paper, focus on the main findings and then just present those for he 12 week follow-up. The rest of the presentation over extends the conclusion that the authors are trying to present.

In essence I don’t feel that the observations require a full and large paper.

1. Abstract: It is unclear that the patients that were non-naïve were on ona. What about inco?

2. Since all of the 4 week data is already published, this is then simply a report of what happened at 12 weeks in terms of efficacy.

3. The issue of who got placebo is totally unclear in the description. Reading the methods, it seems that the non-naïve and the naïve just got abo. No mention is made of who and how many got the placebo from which group.

4. Seems that the non-naïve patients also got placebo? That doesn’t make any sense to me. Were these patients getting efficacy with their toxin? Otr were they selected on the basis that they were not getting efficacy? If they were, they still agreed to get placebo? Is that a reason why many wanted injection earlier in the placebo group?

5. That said, the results say that there was a 40% placebo response?

6. It seems that the two cycles that were selected for ona were not the most recent but two out of 6. Presumably all of the non-naïve patients were already stable on their dose? Did patients have different injections through the 6 cycles prior to the conversion?

7. Assessments: it clearly states the endpoint is 4 weeks. Isn’t this study reporting 12 weeks data? It is not mentioned anywhere in the assessments.

8. When patients were converted, were the sites of injection for ona-versus abo kept the same? Was EMG used for both; i.e. the patients that were getting ona and then got abo had EMG for both? This will potentially make a difference in the outcomes. For example if EMG was not used for Ona and was used for Abo then that could have an outcome effect.

9. Results:

a. It is very odd that there was a 36% drop out upto week 12. Why is this so high? I presume that a number of these patients were getting Ona and then were given placebo? This seems like a poor study design to me if that is the case.

b. A similar concern is regarding the 25 Abo patients requiring earlier toxin injection than 12 weeks. Prior studies have suggested that Abo lasts longer. This casts doubt on such observations

c. Table 1 gives the details that say how many of the different toxins patients were on. 5 patients were on rima. What was the conversion ratio here?

d. It really is also confusing to report week 2 and 4 data that presumably was not the point of this 12 week study. Has this not been reported already in the already published study? For example for the PROs there is no statistics done for the week 12 and the numerical data is all for week 2 and 4. All we are given for the actual week 12 is trends

e. Even with the last known exposure, the average time is 37.8 days and not even close to 12 weeks.

10. Hence it is really quite difficult to agree with the conclusions. The data is very unclear and subjective regarding this endpoint.

11. The 12 week assessment was clearly not an a priori endpoint and that is obvious in the way the data plays out.

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

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PLoS One. 2021 Feb 1;16(2):e0245827. doi: 10.1371/journal.pone.0245827.r002

Author response to Decision Letter 0

21 Dec 2020

Title: Sustained functional benefits after a single set of injections with abobotulinumtoxinA using a 2-mL injection volume in adults with cervical dystonia: 12-week results from a randomized, double-blind, placebo-controlled phase 3b study (PONE-D-19-35636) PLOS ONE

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Author Response: Amendments to style and file have been made as needed.

2. Please amend your current ethics statement to address the following concerns:

a) Did participants provide their written or verbal informed consent to participate in this study?

Author Response: The corresponding paragraph in the Methods section has been expanded as follows: “Subjects were given a full explanation, in lay terms, of the aims of the study, the benefits, potential discomforts and risks of taking part in the study prior to enrollment. A written explanation was also provided and written informed consent was obtained prior to enrollment. The study protocol, subject information leaflet and informed consent document were reviewed and approved by an Institutional Review Board (IRB) prior to commencement of the study. This study was conducted under the provisions of the Declaration of Helsinki, informed consent regulations and in accordance with the International Conference on Harmonisation (ICH) Consolidated Guideline on Good Clinical Practice (GCP) and local site review boards.” (p 7, lines 143-150)

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Author Response: Please see author response to Question 2 above. (p 7, lines 143-150)

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Author Response: Study data include potentially identifying and sensitive information. As a consequence, it is Ipsen policy to share data only with qualified researchers who provide a valid research question. Proposals for research for which research investigators wish to use an Ipsen dataset should be sent to DataSharing@Ipsen.com.

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Author Response: The following information has been added to the ‘Funding/Sponsor’ section: “The funder provided support in the form of salaries for authors [P.M., S.W., and B.R. (formerly)] and support for medical writing of the manuscript, but did not have any additional role in the study design, data collection and analysis, or decision to publish. The specific roles of these authors are articulated in the ‘author contributions’ section.” (p 2, lines 33-37)

Reviewer's Responses to Questions

We thank the reviewers for their feedback and appreciate the opportunity to respond.

Reviewer #1:

1. In this study, the authors present findings on the use of abobotulinum toxin for cervical dystonia when 2ml dilution is used for preparation. The authors present data for outcomes at 12 weeks. They have already published the outcomes at four weeks follow-up. In their analysis of primary outcomes, the authors found that the active group continued to have significant benefits at 12 weeks follow-up compared to placebo. These findings indicate there are longer than expected benefits, as most effects related to botulinum are expected to wane at 12 weeks follow-up.

2. The discussion section is quite underdeveloped. Are these 12 week-outcomes different from those seen with 1ml dilution when evaluated at a similar interval of follow-up? The authors draw comparisons between 1 ml and 2 ml dilution for four weeks' follow-up. The authors should provide scientific insights to understand the value of their findings.

Author Response: The goal of this study was to present the efficacy and safety of aboBoNT-A 500 U/2 mL versus placebo, for up to 12 weeks. These 12-week outcomes are in fact similar to the results seen with the 1 mL dilution, further supporting the claim that using a 500 U/2 mL dilution of aboBoNT-A is just as safe and effective as the 1 mL dilution. These results not only provide scientific insight into the efficacy of aboBoNT-A using a 2 mL dilution, but also the factors associated with increased quality of life.

We have added the following information to the Discussion section: “The weighted overall treatment difference between aboBoNT-A and placebo was –4.8 at Week 12 or at early study withdrawal in the current trial, similar to the adjusted mean difference of –4.3 and -4.1 in the pivotal clinical trials at Week 12.” (p 20, 406-409)

Reviewer #2:

1. Last line of abstract: if most patients were measured at 12 weeks, this would be better as “at 12 weeks” rather than “up to 12 weeks”.

Author Response: We strongly believe that keeping the last line of the abstract as is, “up to 12 weeks” is more accurate due to the fact that patients who withdrew prior to the end of the study visit at week 12 still underwent all procedures required to the Week 12 visit. Therefore, keeping the statement as is captures the fact that the data are representative of all patients, those who completed the Week 12 visit, as well as those who withdrew from the study early.

2. Pg 14- “treatment duration was between 4 and 16 weeks” - wasn’t it a single injection?

Author Response: This study had the following visits, pre-study screening (Day −7 to Day −1, Visit 1), baseline (Day 1, Visit 2), post-treatment follow up (Week 2±2 days, Visit 3 and Week 4±2 days, Visit 4) and study completion or early withdrawal (Week 12±28 days, Visit 5). Patients were given an extra 2-day window for their Visit 3 and 4, and a 28-day window for Visit 5, resulting in the potential for some patients to be enrolled in the study for up to 16 weeks.

We have now expanded the corresponding sentence in the Results section to clarify the reason for treatment duration variation from 4 to 16 weeks, as follows: “Individual treatment duration was between 4 weeks and 16 weeks, due to the study design, which allowed a ±2 day window to complete the Week 2 and 4 visits, and a 28 day window to complete the Week 12 visit.” (p 13, lines 278-280)

Reviewer #3:

This study compares efficacy, patient-reported outcomes, and safety in CD patients receiving 2-ml aboBoNT-A or 2-ml placebo. It is shown that a treatment with 2-ml aboBoNT-A is superior compared to placebo. Besides, the results of the actual study are compared with the results of a previous 1-ml aboBoNT-A study. Since the results are comparable, the administration of aboBoNT-A 500 U using a 2-ml dilution is recommended.

My main criticism of the study is that the design and the limitations of this study have to be discussed.

Comments in detail:

1. Abstract: It is only the dilution (2 ml) mentioned but not the diluted units. Please add so that the concentration is clear.

Author Response: The units using 2-mL dilution have now been added, as follows: “We present efficacy, patient-reported outcomes (PROs), and safety in adults with CD at the last available visit after a single set of abobotulinumtoxinA (aboBoNT-A) injections versus placebo using 500 U, 2-mL injection volume” (p 5, line 88)

2. Introduction: (page 7, line 6): ‘AbobotulinumtoxinA (aboBoNT-A; Dysport®) inhibits acetylcholine release and binds to receptors on peripheral cholinergic nerve endings, and is internalized by receptor-mediated endocytosis. This is followed by cleavage of SNAP25, a protein needed for vesicle fusion to the presynaptic membrane, which is required for synaptosomal release of neurotransmitter into the neuromuscular junction.’

Author Response: The corresponding sentence in the Introduction has now been amended to the following, as suggested by the reviewer: “Botulinum neurotoxin type A binds to receptors on peripheral cholinergic nerve endings and is internalized by receptor-mediated endocytosis.” (p 6, lines 114-116, 118-119)

3. Last sentence of first paragraph: AboBoNT-A is not only approved in the USA but also in Europe for cervical dystonia. This should be also mentioned.

Author Response: The corresponding sentence in the Introduction has now been amended to the following, as suggested by the reviewer: “AboBoNT-A is approved in the United States of America (US) and Europe for the treatment of adults with CD” (p 6, lines 120-121)

4. Second paragraph: This part is a little confusing because only the volume but not the diluted BoNT-A dosis is mentioned. So, the concentration remains unclear. Since there are different vials (300 U and 500 U available) the U per ml has to be mentioned.

Author Response: The sentence has now been expanded as follows: “Initially, the prescribing information in the US specified a 500-unit (U), 1-mL dilution regimen for AboBoNT-A administration; however, feedback obtained from scientific experts, community injectors, and market research studies favored use of a 500 U, 2-mL dilution.” (p 6, lines 123-125)

5. Methods, Treatment (line 7): oba should be changed to ona

Author Response: This correction has been made. (p 9, line 177)

6. Treatment:

a. Regarding your study protocol the onaBoNT-A dosis was converted to aboBoNT-A dosis using a factor of 2.5. This means that the maximal dosis of onaBoNT-A was 200 U since the maximal dosis of aboBoNT-A in your study is limited to 500 U. I would like to know what happened with patients that received more than 200 U of onaBoNT-A (in table 1 it is stated that mean dose of onaBoNT-A was 177+/-34 respectively 176 +/- 42 U. So, in both treatment groups there were onaBoNT-A treated patients that received more than 200 U onaBoNT/A). Was the calculated aboBoNT-A dosis (factor 2.5:1) reduced or have these patients more than 500 U aboBoNT-A received? Please state in the Methods what you have done in such a case.

Author Response: Although the eligibility criteria for this study required that if patients were currently being treated with onaBoNT-A, their dose did not exceed 200 U, 17 patients (10 in the aboBoNT-A group, and 7 in the placebo group) were excluded from the efficacy analysis in the intent-to-treat population due to eligibility criteria violations. As a result, the mean dose of the most recent treatment with onaBoNT-A in both the aboBoNT-A group and in the placebo group does exceed 200 U. These patients, however, were excluded and did not receive the study treatment drug. The footer in Table 1 has been updated to clarify why the mean dose of onaBoNT-A previously received exceeded study eligibility criteria. (p 14, lines 285-287)

b. On the other hand, all BoNT-A native patients received 500 U aboBoNT-A. It is stated that ‘the sites and the dose per site were determined by the investigator according to the standard practice and disease presentation.’ But the total doses were given by the study protocol. I find this difficult because not every patient needs 500 U (corresponding to your patients that were treated with onaBoNT-A before). More explanations to the clinical usage of the fix total dose regiment in your study would be helpful.

Author Response: All BoNT-A naïve patients received 500 U/2-mL of aboBoNT-A based on the results of a previous study (Poewe, et al. 1998) which found the optimal dose of aboBoNT-A for patients with cervical dystonia to be 500 U. We believe this to be a limitation of the study as this dosing procedure differs from that used in clinical practice, where the doses are individualized per patient. Nevertheless, the use of a fixed dose was deemed necessary, as there were no data on which to base dose optimization in BoNT-A naïve patients. This information has now been added to a ‘limitations’ paragraph in the manuscript Discussion section. (p 21, lines 417-426)

7. Results, Figures: there are too many pixels. It is not possible to zoom in and to read the figures easily. Especially in figure 3B: the headings are to bold. It is not possible to read them. Please improve.

Author Response: Resolution of the Figures has been updated for clarity.

8. Discussion, Third paragraph: ‘TWSTERS’ should be changed to ‘TWSTRS’.

Author Response: this correction has now been made. (p 20, line 404)

a. I think it is very important that a paragraph with the limitations of the study is added.

Author Response: The following text has now been added: “Limitations of the study include the use of a fixed dose of aboBoNT-A, which is an important aspect and was done to demonstrate how BoNT-A naïve patients would respond to a standard starting dose. This dosing method differs from that used in clinical practice, where doses are individualized for each patient, and it is likely that many of the participants did not receive the optimal dose in the appropriate muscle. Nonetheless, the use of a fixed dose was necessary, as there were no data available on which to base dose optimization in naïve patients. Additional limitations of this study include the study design allowing patients who were deemed eligible to enter into the open-label extension study between Week 4 and Week 8 (before they reached the planned Week 12 end of the study visit), which resulted in 25 patients withdrawing from the study to enter the open-label extension.” (p 21, lines 417-426)

We agree that a direct comparison of a 1 mL and 2 mL dilution to placebo would be interesting, and we hope based on the results of our study, it can be done in the future.

Although the efficacy of aboBoNT-A versus placebo is not new, safety and efficacy of a single 500 U/2 mL dilution injection of aboBoNT-A for up to 12 weeks has not yet been demonstrated, as it has been with a 1 mL dilution. As there had been a lack of scientific data and literature regarding the use of a 500 U/2 mL dilution of aboBoNT-A versus placebo, we believe that the results of this study support the efficacy and safety of using a 500 U/2 mL dilution to better meet patient needs.

Feedback received from scientific experts, community injectors, and market research studies favored the use of a 2 mL dilution, specifically. This was most likely related to prior familiarity in using comparable volumes with other approved toxins in the US. In a study conducted in Europe (Yun, et al. 2015), a 2 mL dilution of aboBoNT-A was used to demonstrate the efficacy and safety of aboBoNT-A in comparison with onaBoNT-A. Therefore, in this clinical study, the majority of enrolled patients had previously been treated with onaBoNT-A, in order to reflect the real world clinical scenario in the US.

Although different indications may allow larger dilution volumes, the recommended concentration range for cervical dystonia is 25 or 50 U/0.1 mL, with a aboBoNT-A dose of 500 to 1000 U. (No changes to the manuscript text)

In general, this is a complete and well written about sustained abobotulinumtoxinA efficacy in cervical dystonia.

Author Response: We thank Reviewer #4 for this review and comments.

Reviewer #5:

Overall impression: I found this paper unclear to read. It is also too long for a very simple conclusion that a 2ml dilution also works. I am not convinced that this small conclusion requires a rehash of the entire study that has already been published. In my mind this is a short report only. Much of the actual 12week data is presented subjectively with very mixed if any statistics, lots of drops outs, and early treatments etc and is truly hard to interpret. I would strongly suggest the authors to substantially shorten the paper, focus on the main findings and then just present those for the 12 week follow-up. The rest of the presentation over extends the conclusion that the authors are trying to present. In essence I don’t feel that the observations require a full and large paper.

a. Abstract: It is unclear that the patients that were non-naïve were on ona. What about inco?

Author Response: As previously published in the primary manuscript, eligible patients who were non-naïve and currently undergoing treatment with onaBoNT-A (receiving a total dose of 100-200 U, and ≤60 U in the sternocleidomastoid muscle (SCM), since the last injection cycle, could have received any other formulation of BoNT-A prior to study enrollment, as long as they had a satisfactory clinical response to the last two sequential cycles with onaBoNT-A in the past 18 months, or had not received onaBoNT-A for at least 12 weeks. (No changes to manuscript text)

b. Since all of the 4-week data is already published, this is then simply a report of what happened at 12 weeks in terms of efficacy.

Author Response: This study presents the efficacy, patient-reported outcomes, and safety results at the last available visit, after a single injection of aboBoNT-A 500 U/2 mL dilution versus placebo, further supporting the results of the primary publication.

As the methodology and primary efficacy endpoints have already been published, we have included only the necessary data.

The study rationale was based on previously published data (Yun, et al, 2015) which demonstrated that aboBoNT-A at a conversion ratio of 2.5:1 was not inferior in comparison to onaBoNT-A, utilizing a 2 mL dilution. (No changes to manuscript text)

c. The issue of who got placebo is totally unclear in the description. Reading the methods, it seems that the non-naïve and the naïve just got abo. No mention is made of who and how many got the placebo from which group.

Author Response: Patients were randomized in blocks, based on a computer-generated randomization list. List A stratified patients who were toxin naïve or non-naïve at baseline with a 2:1 ratio of aboBoNT-A:placebo. List B was produced on a 1:1 basis (aboBoNT-A:placebo) of treatment numbers, which were specified on the treatment packs, to be dispatched to the sites in order to dispense the drug. (No changes to manuscript text)

d. Seems that the non-naïve patients also got placebo? That doesn’t make any sense to me. Were these patients getting efficacy with their toxin? Otr were they selected on the basis that they were not getting efficacy? If they were, they still agreed to get placebo? Is that a reason why many wanted injection earlier in the placebo group?

Author Response: Please see the above response (Reviewer 5, comment “c”).

e. That said, the results say that there was a 40% placebo response?

Author Response: Although 40% of patients in the placebo group reported being at least somewhat satisfied with the medication, approximately twice as many patients in the aboBoNT-A group were at least somewhat satisfied across time points (Week 2: abobotulinumtoxinA, 64.8%, vs placebo, 32.5%; Week 4: abobotulinumtoxinA, 65.1%, vs placebo, 35.6%; last available: abobotulinumtoxinA, 64.0%, vs placebo, 40.0%). (No changes to manuscript text)

f. It seems that the two cycles that were selected for ona were not the most recent but two out of 6. Presumably all of the non-naïve patients were already stable on their dose? Did patients have different injections through the 6 cycles prior to the conversion?

Author Response: Eligible non-naïve patients currently treated with onaBoNT-A at a total dosing range of 100 U to 200 U and ≤60 U in the SCM at the last injection cycle and had a satisfactory treatment response in the enrolling investigator's judgment during the last two sequential cycles of onaBoNT-A within the past 18 months for the treatment of CD, and a minimum of 12 weeks since the last onaBoNT-A injection. (No changes to manuscript text)

g. Assessments: it clearly states the endpoint is 4 weeks. Isn’t this study reporting 12 weeks data? It is not mentioned anywhere in the assessments.

Author Response: This statement was in the Statistical analysis section. It has now been added to the study design. “The study had the following visits: prestudy screening (Day -7 to Day -1, Visit 1), baseline (Day 1, Visit 2; this visit could have occurred on the same day as the screening visit at the discretion of the investigator), post-treatment follow up (Week 2±2 days, Visit 3 and Week 4±2 days, Visit 4) and study completion or early withdrawal (Week 12+28 days, Visit 5). All subjects who had completed the Week 12 visit were considered to have completed the study.”

Also added to assessments “Tertiary efficacy endpoints included assessments of the change from baseline at Week 12.” (Page 9, lines 190-191)

h. When patients were converted, were the sites of injection for ona-versus abo kept the same? Was EMG used for both; i.e. the patients that were getting ona and then got abo had EMG for both? This will potentially make a difference in the outcomes. For example if EMG was not used for Ona and was used for Abo then that could have an outcome effect.

Author Response: Study drug injections in non-naïve patients were given into previously injected muscles (Study Methods, p 8, 157-161). Electromyography-guided injections were allowed, at the preference of the investigator at each site (Treatment, p 8, lines 174-175). (No changes to manuscript text)

i. Results:

a. It is very odd that there was a 36% drop out up to week 12. Why is this so high? I presume that a number of these patients were getting Ona and then were given placebo? This seems like a poor study design to me if that is the case.

b. A similar concern is regarding the 25 Abo patients requiring earlier toxin injection than 12 weeks. Prior studies have suggested that Abo lasts longer. This casts doubt on such observations

Author Response: Reasons for patient exclusions from the study were eligibility criteria violations, randomization/treatment allocation process violations, study treatment administration violation, time window violations, and TWSTRS data not available, or the patient was not treated.

Due to the study design, eligible patients were allowed to enter into the open-label extension study between Week 4 and Week 8, and prior to the Week 12 study conclusion visit. As a result, 47 patients (25 in aboBoNT-A, 22 in placebo) elected to enter the open-label extension portion of the study prior to Week 12, with 33 patients entering at Week 4. This information is already included in the manuscript. (No changes to manuscript text)

j. Table 1 gives the details that say how many of the different toxins patients were on. 5 patients were on rima. What was the conversion ratio here?

Author Response: Although patients receiving other formulations were included, the conversion ratio used was aboBoNT-A:onaBoNT-A. Table 1 summarizes the previous BoNT-A treatments the patients had received. (No changes to manuscript text)

k. It really is also confusing to report week 2 and 4 data that presumably was not the point of this 12 week study. Has this not been reported already in the already published study? For example for the PROs there is no statistics done for the week 12 and the numerical data is all for week 2 and 4. All we are given for the actual week 12 is trends

Author Response: Week 2 and Week 4 data are included to show the efficacy and safety, and patient-level benefits of aboBoNT-A as early as Week 2 and up to Week 12. (No changes to manuscript text)

l. Even with the last known exposure, the average time is 37.8 days and not even close to 12 weeks.

Author Response: The average study exposure, as stated in Table 2, for the total intent-to-treat population was 62.6 (SD 37.8) days, with a median of 85.0 days. In the aboBoNT-A group, the average study exposure was 67.7 (SD 38.4). (No changes to manuscript text)

m. Hence it is really quite difficult to agree with the conclusions. The data is very unclear and subjective regarding this endpoint.

Author Response: These findings do in fact support our conclusions in terms of the efficacy of aboBoNT-A having longer than expected benefits. (No changes to manuscript text)

n. The 12 week assessment was clearly not an a priori endpoint and that is obvious in the way the data plays out.

Author Response: A hierarchical testing procedure combined the primary and ranked secondary efficacy endpoints together with type I error controlled at a level of 5%. The superiority of aboBoNT-A to placebo on the rank-1 secondary efficacy endpoint was tested using the same method as the one used for the primary efficacy endpoint. The hierarchical testing procedure was stopped if there was no statistically significant treatment effect on the current efficacy endpoint, otherwise the next step was performed. Exploratory analysis was performed for each of the tertiary efficacy endpoints. Summary statistics were summarized by treatment group and not compared by formal statistical testing. Confidence intervals were estimated for some endpoints to characterize the full clinical effect but no statistical conclusions were drawn. (No changes to manuscript text)

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

Reviewer #5: No

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PLoS One. doi: 10.1371/journal.pone.0245827.r003

Decision Letter 1

Mandar S Jog

11 Jan 2021

PONE-D-19-35636R1

Dear Dr. Wietek,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Mandar S Jog

Guest Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0245827.r004

Acceptance letter

Mandar S Jog

19 Jan 2021

PONE-D-19-35636R1

Dear Dr. Wietek:

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Associated Data

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Supplementary Materials

S1 Fig. Study site locations.

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Data Availability Statement

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PERMALINK

Sustained functional benefits after a single set of injections with abobotulinumtoxinA using a 2-mL injection volume in adults with cervical dystonia: 12-week results from a randomized, double-blind, placebo-controlled phase 3b study

Atul T Patel

Mark F Lew

Khashayar Dashtipour

Stuart Isaacson

Robert A Hauser

William Ondo

Pascal Maisonobe

Stefan Wietek

Bruce Rubin

Allison Brashear

Roles

Abstract

Introduction

Methods

Study design

Patients

Treatment

Assessments

Sample size

Randomization

Blinding

Statistical analysis

Data sharing

Results

Patient disposition and study exposure

Table 1. Baseline demographics and characteristics (ITT populations).

Fig 1. CONSORT diagram.

Table 2. Patient (ITT) study exposure for all patients and patients who withdrew from the study.

TWSTRS

Fig 2. Mean TWSTRS total score.

Patient-reported outcomes

Fig 3.

Satisfaction

Safety

Table 3. Treatment-emergent adverse events in the safety set.

Discussion/conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Nancy Beam

Roles

Author response to Decision Letter 0

Decision Letter 1

Mandar S Jog

Roles

Acceptance letter

Mandar S Jog

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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