BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from

PEER REVIEW HISTORY

BMJ Open publishes all reviews undertaken for accepted manuscripts. Reviewers are asked to complete a checklist review form (http://bmjopen.bmj.com/site/about/resources/checklist.pdf) and are provided with free text boxes to elaborate on their assessment. These free text comments are reproduced below.

ARTICLE DETAILS

TITLE (PROVISIONAL) Effectiveness of pharmacological treatments in Duchenne muscular dystrophy: a protocol for a systematic review and meta- analysis AUTHORS Pascual Morena, Carlos; Martinez-Vizcaino, Vicente; Álvarez- Bueno, Celia; Fernández Rodríguez, Ruben; Jiménez López, Estela; Torres-Costoso, Ana Isabel; Cavero-Redondo, Iván

VERSION 1 - REVIEW

REVIEWER Doris G. Leung Kennedy Krieger Institute, USA REVIEW RETURNED 10-Mar-2019

GENERAL COMMENTS Thank you for the opportunity to review this protocol. The authors propose a systematic review and meta-analysis of pharmacologic and nutraceutical interventions in Duchenne muscular dystrophy.

The topic is a timely one, as there are multiple ongoing clinical http://bmjopen.bmj.com/ trials in the Duchenne population and the body of literature on this topic is both dynamic and controversial. I have a number of questions, comments, and recommendations, and these are delineated below.

Major comments

The search terms are divided into 3 themes. The first defines the on September 23, 2021 by guest. Protected copyright. disease to be DMD, the third specifies that the studies included should be interventional trials. I am concerned that the second theme, which lists therapies that have been tested in DMD, is too restrictive in several ways: 1) This search would effectively exclude many relevant recent pharmaceutical trials in DMD, including those of myostatin inhibitors, idebenone, phosphodiesterase inhibitors, NF-kappaB inhibitors, utrophin modulators, metformin, and others. The search would also exclude a number of older trials of drugs like perindopril and albuterol. 2) The search terms also exclude trials of corticosteroids. As corticosteroids are the only drugs that have been shown to improve muscle strength across the Duchenne population, these trials are extremely important within the field. So much so that there have been new trial results published on corticosteroids as recently as 2016 (Griggs R. et al, Neurology, 2016) as well as ongoing trials of new classes of steroids like vamorolone. 3) By naming individual drugs (like eteplirsen and ataluren) without their larger class (i.e., exon-skipping, nonsense readthrough), the BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from investigators risk missing new drugs in those classes, such as golodirsen and casimersen, which are currently being tested in trials. The authors seem to address this issue in part by stating in the methodology section that they are specifically interested in analyzing drugs acting at the ribosomal or epigenetic level, but if that is the case there doesn’t seem to be a compelling reason to include studies on nutraceuticals at all, since the mechanisms of action for nutraceuticals are probably not occurring at the ribosomal or epigenetic level. I suspect that restricting the scope of the search this much will exclude too many of the trials that are of the greatest interest to the Duchenne population, and in doing so will curtail any real impact such a review may have on the field.

The authors do not describe any methodology for searching gray literature. In the field of DMD in particular, it would be useful to search outside of major databases, as trial results may not be published as manuscripts, but instead reported in press announcements, conference proceedings/abstracts, websites, and submissions to regulatory agencies. I would also recommend searching trial registries, like clinicaltrials.gov and the EU Clinical Trials Register. Additional trials and results may also be identified through Duchenne advocacy groups, patient registries, and fundraising organizations.

Minor comments

Introduction/Background: “...losing ambulation and becoming dependent on a wheelchair at 12 years of age.” is oversimplifying the case. The mean age of loss of ambulation is 12, but a significant proportion of DMD patients are maintaining ambulation until 16 or later, likely due to mutation type as well as improvements in management. Similarly, stating that death occurs “by the second to third decade of life” does not reflect the most current population data that shows that the average life http://bmjopen.bmj.com/ expectancy is closer to 30 years and that a growing number of DMD patients are surviving into the 4th and 5th decades of life.

Introduction/State of the art: “This led to the synthesis of ataluren, more effective than gentamicin, producing nonsense mutations that cause a premature stop codon…” This sentence is awkwardly worded. Neither gentamicin nor ataluren produce nonsense mutations; both are designed to promote readthrough of the existing nonsense mutations. on September 23, 2021 by guest. Protected copyright.

Methodology: “Type of study design: clinical trials using or not random allocation…” is also confusing. Consider using “clinical trials without restriction based on allocation or blinding strategy.”

The listing for this systematic review on PROSPERO reports that articles not written in English or Spanish will be excluded, but the submitted manuscript states that no language exclusion will be applied. Please reconcile.

Methodology/Type of outcome: omission of exon 51 is listed under histological changes, but detection of exon 51 skipping is not measured with histological techniques.

Consider including “dystrophin” and/or “dystrophinopathy” as search terms. Dystrophinopathy is increasingly being used to BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from describe the spectrum of diseases that include Duchenne, Becker, and intermediate disease forms.

The search terms include “RTC” and “non-RTC.” These should probably be RCT for “randomized controlled trials.”

The search terms will also exclude: 1) trials of gene therapy, stem cell treatments, and gene editing; 2) trials that do not have skeletal muscle strength as their primary outcomes (i.e. cardiac trials of eplerenone, ACE inhibitors, or pulmonary function trials). Since these interventions (the first group in particular) are of significant interest to the muscular dystrophy field, it would be appropriate to state that they are specifically being excluded from the review and perhaps to offer a rationale for this decision.

Methodology/Evaluation of the quality of the studies: risk of bias: “Additionally, due to the severity of the disease and how little susceptible it is to misinterpretation of data…” It is not clear what this sentence is trying to communicate. Are they saying that this data is less susceptible to bias or more?

Methodology/Evaluation of the quality of the studies: risk of bias: It would be useful to include a description of how the risk of bias assessments will be incorporated into the analysis. For instance, will the analysis be restricted to studies with low risk of bias or will data be stratified by risk of bias?

Methodology/Sensitivity analysis: “These will be performed to prove that the findings from the meta-analysis do not depend on arbitrary or unclear decisions.” This sentence needs clarification. This type of sensitivity analysis doesn’t really exclude the possibility of arbitrary or unclear decisions, but rather assesses whether or not the data is excessively skewed by influential data points. http://bmjopen.bmj.com/

Discussion: “Conversely, other physical tests (i.e., tests that measure muscle strength or the ability to climb and descend steps) are more elaborate and do not evaluate physical resistance.” Please clarify: how are the stair-climbing tests “more elaborate” than the 6-minute walk, and how are they not dependent on physical resistance (in this case defined as cardiorespiratory fitness)? on September 23, 2021 by guest. Protected copyright. Table 2: A number of trials in DMD utilize crossover study designs and open-label extension studies where participants who were previously in placebo groups eventually are given the study drug. How do the authors intend to present data from these study designs?

Table 2: The authors state that they will include information on the year and country in which the study takes place, but these variables are not included in the table.

Figure 1: Consider excluding this figure. In the absence of the numbers of records, it doesn’t add any specific information to the manuscript. It would be sufficient to reference or include a link to the PRISMA flow diagram template.

BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from

REVIEWER Fredrik Sjövall Lund University, Institution of Clinical Sciences REVIEW RETURNED 04-Apr-2019

GENERAL COMMENTS Thank you for letting me review the manuscript by Morena et al where they describe a protocol for a systematic review of current treatment of Duchennes muscle dystrophy. I have some comments In general, I believe that the language in the manuscript needs revising. There are several parts that are difficult to understand which is caused by bad formulation or wording. I believe that the whole study design is quite vague and broad more closing towards a narrative review and the authors should be more There is no hypotheis on what the authors believe would be an important treatment effect. It is also difficult to assess what type of trials that is going to be included. The authors state ”clinical trials using or not random allocation or blinding strategies.” It is not clear what the authors mean by this. Also, in the ”Intervention” section of the PICO they state ”trials that study the influence of nutraceuticals on some variable of interest” The variables of interest has to be defined a priori and not made up as the authors are reading the literature. Will there be used a standardised data extraction form? This should then be stated and if not, is should be developed, and stated.

VERSION 1 – AUTHOR RESPONSE

Reviewer #1 http://bmjopen.bmj.com/ Comments:

 The search terms are divided into 3 themes. The first defines the disease to be DMD, the third specifies that the studies included should be interventional trials. I am concerned that the second theme, which lists therapies that have been tested in DMD, is too restrictive in several ways: 1) This search would effectively exclude many relevant recent pharmaceutical trials in DMD, including those of myostatin inhibitors, idebenone, phosphodiesterase inhibitors, NF- kappaB inhibitors, utrophin modulators, metformin, and others. The search would also on September 23, 2021 by guest. Protected copyright. exclude a number of older trials of drugs like perindopril and albuterol. 2) The search terms also exclude trials of corticosteroids. As corticosteroids are the only drugs that have been shown to improve muscle strength across the Duchenne population, these trials are extremely important within the field. So much so that there have been new trial results published on corticosteroids as recently as 2016 (Griggs R. et al, Neurology, 2016) as well as ongoing trials of new classes of steroids like vamorolone. 3) By naming individual drugs (like eteplirsen and ataluren) without their larger class (i.e., exon-skipping, nonsense readthrough), the investigators risk missing new drugs in those classes, such as golodirsen and casimersen, which are currently being tested in trials. The authors seem to address this issue in part by stating in the methodology section that they are specifically interested in analyzing drugs acting at the ribosomal or epigenetic level, but if that is the case there doesn’t seem to be a compelling reason to include studies on nutraceuticals at all, since the mechanisms of action for nutraceuticals are probably not occurring at the ribosomal or epigenetic level. I suspect that restricting the scope of the search this much will exclude too many of the trials that are of BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from

the greatest interest to the Duchenne population, and in doing so will curtail any real impact such a review may have on the field.

Authors

The reviewer’s comment seems judicious. We have modified the search strategy including all drugs and pharmacological groups relevant in the treatment of Duchenne Muscular Dystrophy. We have also eliminated the group of nutraceuticals because it seems not appropriate to include them in this systematic review (Page 5):

“The search strategy terms will be the following: (‘dystrophy’ OR ‘duchenne’ OR ‘dmd’ OR ‘dystrophinopathy’) AND (‘nonsense readthrough’ OR ‘gentamicin’ OR ‘ataluren’ OR ‘exon skipping’ OR ‘antisense oligonucleotides’ OR ‘eteplirsen’ OR ‘drisapersen’ OR ‘golodirsen’ OR ‘casimersen’ OR ‘histone deacetylase inhibitor’ OR ‘myostatin inhibitor’ OR ‘phosphodiesterase inhibitor’ OR ‘nf-κb inhibitor’ OR ‘utrophin modulator’ OR ‘immunosuppressant’ OR ‘glucocorticoids’ OR ‘prednisone’ OR ‘deflazacort’ OR ‘vamorolone’ OR ‘idebenone’ OR ‘beta agonist’ OR ‘angiotensin-converting-enzyme inhibitor’ OR ‘angiotensin II receptor blocker’ OR ‘beta blocker’ OR ‘vasodilator’ OR ‘diuretic’ OR ‘gene therapy’ OR ‘gene editing’) AND (‘‘randomized clinical trial’ OR ‘randomized controlled trial’ OR ‘clinical trial’).”

 The authors do not describe any methodology for searching gray literature. In the field of DMD in particular, it would be useful to search outside of major databases, as trial results may not be published as manuscripts, but instead reported in press announcements, conference proceedings/abstracts, websites, and submissions to regulatory agencies. I would also recommend searching trial registries, like clinicaltrials.gov and the EU Clinical Trials Register. Additional trials and results may also be identified through Duchenne advocacy groups, patient registries, and fundraising organizations.

Authors:

As suggested, we have included the search description the most relevant gray literature databases, http://bmjopen.bmj.com/ as well as clinical trial records ClinicalTrials.gov and EudraCT (Page 5):

“Searches for unpublished studies will be conducted at: OPEN GRAY, ProQuest dissertations & Thesis Global, Theseo, Networked digital library of theses and dissertations (NDLTD), and Google Scholar. A search of ClinicalTrials.gov and EudraCT clinical trial records will also be conducted.”

 Introduction/Background: “...losing ambulation and becoming dependent on a wheelchair at

12 years of age.” is oversimplifying the case. The mean age of loss of ambulation is 12, but a on September 23, 2021 by guest. Protected copyright. significant proportion of DMD patients are maintaining ambulation until 16 or later, likely due to mutation type as well as improvements in management. Similarly, stating that death occurs “by the second to third decade of life” does not reflect the most current population data that shows that the average life expectancy is closer to 30 years and that a growing number of DMD patients are surviving into the 4th and 5th decades of life.

Authors

The reviewer’s comment seems judicious. Thus, we have modified this paragraph in Introduction/Background section and included the causes of the improvement in the prognosis of the disease, as well as potential causes of interindividual differences in its progression (Page 3):

“[…] However, improvements in the management of DMD has delayed the appearance of these events. The use of glucocorticoids and physiotherapy delays the loss of strength and ambulation5,6; early pharmacological therapy for heart failure improves prognosis and survival7; surgery for scoliosis, respiratory physiotherapy and the use of non-invasive ventilation have enhanced lung capacity, BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from hypercapnia and respiratory failure, increasing survival8,9,10. Also, a high interindividual intrinsic variability in the progression of the disease due to the type of mutation in the dystrophin gene, or to polymorphisms in other genes, is characteristic11”

 Introduction/State of the art: “This led to the synthesis of ataluren, more effective than gentamicin, producing nonsense mutations that cause a premature stop codon…” This sentence is awkwardly worded. Neither gentamicin nor ataluren produce nonsense mutations; both are designed to promote readthrough of the existing nonsense mutations.

Authors:

Thank you for reviewer’s comment. We have modified the paragraph improving the wording of the explanation as follows (Page 3):

“These lines of research are mostly focused on the partial production of dystrophin, such as happens in the muscular dystrophy of Becker (BMD), which has a less severe prognosis than DMD because the reading frame of the gene is not affected, and a partially functional dystrophin is produced14. Almost two decades ago, gentamicin was found to cause, in a small percentage of patients, some production of dystrophin forcing the reading of premature stop codons, introducing other tRNAs and being able to continue the synthesis of protein15. The ataluren, which was developed with this same mechanism of action, have demonstrated to be more effective than gentamicin16.”

 Methodology: “Type of study design: clinical trials using or not random allocation…” is also confusing. Consider using “clinical trials without restriction based on allocation or blinding strategy.”

Authors:

Thank you for reviewer’s comment. As suggested, we have modified the sentence as follows (Page 4):

“Type of study design: clinical trials without restriction based on allocation or blinding strategy.” http://bmjopen.bmj.com/

 The listing for this systematic review on PROSPERO reports that articles not written in English or Spanish will be excluded, but the submitted manuscript states that no language exclusion will be applied. Please reconcile.

Authors:

We apologize for the mistake; thus, we have modified this inclusion criteria in abstract and on September 23, 2021 by guest. Protected copyright. methodology sections as follows:

“Search will be conducted in English and Spanish.”

 Methodology/Type of outcome: omission of exon 51 is listed under histological changes, but detection of exon 51 skipping is not measured with histological techniques.

Authors:

We really appreciate the reviewer’s thoughtful comment. Thus, we have modified this information in Type of outcome sub-section, highlighting the method of detecting the omission of exon 51 (Page 5).

“v) molecular changes: detection of omission of exon 51 by reverse transcription polymerase chain reaction (RT-PCR)” BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from

 Consider including “dystrophin” and/or “dystrophinopathy” as search terms. Dystrophinopathy is increasingly being used to describe the spectrum of diseases that include Duchenne, Becker, and intermediate disease forms.

Authors:

Thank you. We have included these terms in the search strategy.

 The search terms include “RTC” and “non-RTC.” These should probably be RCT for “randomized controlled trials.”

Authors:

Done. Thank you. We have included ‘randomized clinical trial’, ‘randomized controlled trial’ and ‘clinical trial’.

 The search terms will also exclude: 1) trials of gene therapy, stem cell treatments, and gene editing; 2) trials that do not have skeletal muscle strength as their primary outcomes (i.e. cardiac trials of eplerenone, ACE inhibitors, or pulmonary function trials). Since these interventions (the first group in particular) are of significant interest to the muscular dystrophy field, it would be appropriate to state that they are specifically being excluded from the review and perhaps to offer a rationale for this decision.

Authors:

The reviewer’s comment seems judicious. We have modified the search strategy including terms for gene therapy and treatments for the complications associated with the disease, as well as its rationale in the introduction section.

Introduction (Page 4):

“In recent years, the interest in the potential of gene editing has increased, since new therapeutic http://bmjopen.bmj.com/ strategic such as inserting dystrophin functional genes through adenovirus,21 repairing the affected gene using the CRISPR/Cas9 technique,22 or transplanting muscle stem cells to the affected muscles22,23 have proven some effectiveness.”

Methodology (Page 5):

“The search strategy terms will be the following: (‘dystrophy’ OR ‘duchenne’ OR ‘dmd’ OR

‘dystrophinopathy’) AND (‘nonsense readthrough’ OR ‘gentamicin’ OR ‘ataluren’ OR ‘exon skipping’ on September 23, 2021 by guest. Protected copyright. OR ‘antisense oligonucleotides’ OR ‘eteplirsen’ OR ‘drisapersen’ OR ‘golodirsen’ OR ‘casimersen’ OR ‘histone deacetylase inhibitor’ OR ‘myostatin inhibitor’ OR ‘phosphodiesterase inhibitor’ OR ‘nf-κb inhibitor’ OR ‘utrophin modulator’ OR ‘immunosuppressant’ OR ‘glucocorticoids’ OR ‘prednisone’ OR ‘deflazacort’ OR ‘vamorolone’ OR ‘idebenone’ OR ‘beta agonist’ OR ‘angiotensin-converting-enzyme inhibitor’ OR ‘angiotensin II receptor blocker’ OR ‘beta blocker’ OR ‘vasodilator’ OR ‘diuretic’ OR ‘gene therapy’ OR ‘gene editing’) AND (‘‘randomized clinical trial’ OR ‘randomized controlled trial’ OR ‘clinical trial’).”

 Methodology/Evaluation of the quality of the studies: risk of bias: “Additionally, due to the severity of the disease and how little susceptible it is to misinterpretation of data…” It is not clear what this sentence is trying to communicate. Are they saying that this data is less susceptible to bias or more?

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Authors:

We really appreciate the reviewer’s thoughtful comment. Because removing considerations regarding the severity of the disease the risk of bias will be more rigorous we have decided to delete this sentence.

 Methodology/Evaluation of the quality of the studies: risk of bias: It would be useful to include a description of how the risk of bias assessments will be incorporated into the analysis. For instance, will the analysis be restricted to studies with low risk of bias or will data be stratified by risk of bias?

Authors:

The reviewer’s comment seems appropriate. As suggested, we have modified the paragraph including a sentence stating that those studies assessed as high risk of bias will be removed.

In the Evaluation of the quality of the studies: Risk of bias section (Page 6):

“The risk of bias assessment will be conducted using the RoB 2.0 tool developed by the Cochrane Collaboration. This version includes five domains: bias arising from the randomization process, bias due to deviations from intended interventions, bias due to missing outcome data, bias in measurement of the outcome, bias in selection of the reported result. Each domain is scored as high risk, some concerns or low risk. A sixth domain, overall bias, will have a low risk result if the rest of the domains are of low risk; some concerns if there are some domain assessed with some concerns; and high risk if there is a domain or more with high risk of bias. Studies scoring as high risk will be removed of the meta-analysis.”

In the Analysis by subgroups section (Page 7):

“[…] In addition, a subgroup analysis will be conducted depending on the result obtained in the assessment of the risk of bias. Thus, two subgroups will be considered: without domains with a high risk of bias; and with one or more domains with a high risk of bias.” http://bmjopen.bmj.com/

 Methodology/Sensitivity analysis: “These will be performed to prove that the findings from the meta-analysis do not depend on arbitrary or unclear decisions.” This sentence needs clarification. This type of sensitivity analysis doesn’t really exclude the possibility of arbitrary or unclear decisions, but rather assesses whether or not the data is excessively skewed by influential data points.

Authors: on September 23, 2021 by guest. Protected copyright.

Thank you. As suggested, we have modified the sentence in order to clarify the sensitivity analysis section as follows (Page 7):

“[…] These will be performed to examine the potential influence of each study in the pooled estimates.”

 Discussion: “Conversely, other physical tests (i.e., tests that measure muscle strength or the ability to climb and descend steps) are more elaborate and do not evaluate physical resistance.” Please clarify: how are the stair-climbing tests “more elaborate” than the 6-minute walk, and how are they not dependent on physical resistance (in this case defined as cardiorespiratory fitness)?

Authors: BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from

Thank you for reviewer’s comment. We have deleted that sentence in order to prevent from misinterpretation, and because the sentence did not add any valuable information.

 Table 2: A number of trials in DMD utilize crossover study designs and open-label extension studies where participants who were previously in placebo groups eventually are given the study drug. How do the authors intend to present data from these study designs?

Authors:

This is really a good point. We will display the results up to the crossing point, and the results at the end of the trial, in table 2 and in the narrative synthesis. In the meta-analysis, we will prioritize data collected at crossing point (Page 6):

“All trials included in the meta-analyzes will be controlled trials, compared to placebo, non- intervention, and exceptionally, matched historical cohort. In cross-over trials, we will preferably use the results at the end of the first period. A narrative synthesis of the data will be presented for the remaining outcomes.”

 Table 2: The authors state that they will include information on the year and country in which the study takes place, but these variables are not included in the table.

Authors:

Thank you for reviewer’s comment. As suggested, we have included this information in Table 1.

 Figure 1: Consider excluding this figure. In the absence of the numbers of records, it doesn’t add any specific information to the manuscript. It would be sufficient to reference or include a link to the PRISMA flow diagram template.

Authors:

Done. Thank you. http://bmjopen.bmj.com/

Reviewer #2

Comments

 In general, I believe that the language in the manuscript needs revising. There are several on September 23, 2021 by guest. Protected copyright. parts that are difficult to understand which is caused by bad formulation or wording.

Authors:

Thank you for reviewer’s comment. The manuscript has been reviewed by a scientific expert professional English language to enhance wording.

 I believe that the whole study design is quite vague and broad more closing towards a narrative review and the authors should be more

Authors:

We really appreciate the reviewer’s thoughtful comment. We have included several changes in the manuscript, particularly in the "Data synthesis" section, and included a new table (Table 2), in order to present an explicit and replicable methodology for the systematic review and meta-analysis.

 There is no hypothesis on what the authors believe would be an important treatment effect. BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from

Authors:

The reviewer’s comment seems judicious. As suggested, we have included a paragraph in the Introduction section describing the rationale for this systematic review as follows (Page 4):

“Despite numerous trials testing new treatments for DMD have recently been conducted, there is a lack of evidence about the effectiveness of these treatments on delaying the progression of the disease. Furthermore, whether the early beginning of the management of comorbidities improves the functional status and the prognosis of the patients, as well as which are the most effective drugs for to treat these complications, is an important issue that should be clarified.”

 It is also difficult to assess what type of trials that is going to be included. The authors state ”clinical trials using or not random allocation or blinding strategies.”. It is not clear what the authors mean by this.

Authors:

Thank you for reviewer’s comment. We have modified the sentence as follows (Page 4).

“Type of study design: clinical trials without restriction based on allocation or blinding strategy.”

 Also, in the ”Intervention” section of the PICO they state ”trials that study the influence of nutraceuticals on some variable of interest” The variables of interest has to be defined a priori and not made up as the authors are reading the literature.

Authors:

We really appreciate the reviewer’s thoughtful comment. We have decided to remove the group of nutraceuticals because its study is not considered appropriate in this systematic review.

 Will there be used a standardized data extraction form? This should then be stated and if not, is should be developed, and stated. http://bmjopen.bmj.com/

Authors:

Thank you for reviewer’s comment. We have modified the paragraph of "Data synthesis" and we have added Table 2, which includes the standardized extraction of data for the most relevant outcomes as follows (Page 6 and Page 15):

“Data synthesis on September 23, 2021 by guest. Protected copyright.

Forest plots will be used to indicate the effect size, with its 95% confidence interval (95% CI), for the effect of interventions in: the change of 6MWD, measured as difference in meters ± SD; the change in TFT (up 4 stairs, down 4 stairs, run 10 meters, supine to stand), measured in seconds ± SD; the change in NSAA ± SD (Table 2). For the conducting of each meta-analysis, at least five trials that determine the change in the outcome for a specific drug will be needed. All trials included in the meta- analyzes will be controlled trials, compared to placebo, non-intervention, and exceptionally, matched historical cohort. In cross-over trials, we will preferably use the results at the end of the first period. A narrative synthesis of the data will be presented for the remaining outcomes.”

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“Table 2. Data collection form for the variables included in meta-analysis”

Reference Drug Subgro 6MWD Up 4 stairs Down 4 Run 10 m Supine to NSAA up stairs stand Δx̄ (m SD(m Δx̄ (s) SD(s Δx̄ (s) SD(s Δx̄ (s) SD(s Δx̄ (s) SD(s Δ S ) ) ) ) ) ) x̄ D Authors, Drug By age ------year used or basal 6MWD 6MWD: Six-minute walking distance; NSAA: North Star Ambulatory Assessment; Δx̄ : Difference between intervention/control groups; SD: Deviation Standard.

VERSION 2 – REVIEW

REVIEWER Doris G. Leung Kennedy Krieger Institute, USA REVIEW RETURNED 08-Jun-2019

GENERAL COMMENTS The majority of the concerns that I had with the original manuscript have been appropriately addressed. I do have some concerns about the revised version, which I have described in detail below:

Article summary: The authors state that the study will, “enhance the transparency of research, reducing publication bias, preventing selective publication and selective reporting of research outcomes.” I would consider rephrasing this. A systematic review doesn’t actively reduce publication bias or prevent selective

publication because it only evaluates articles after they have http://bmjopen.bmj.com/ already been published. The methodology that the authors describe can identify publication bias or selectivity in reporting, but it doesn’t correct these problems.

Article summary: “The publication of this protocol for systematic reviews and meta-analysis prevents from unnecessary duplication of research by describing which outcomes and selection criteria of

trials are planned to be considered in the systematic review and on September 23, 2021 by guest. Protected copyright. meta-analysis.” I’m not sure that publishing the protocol will actually prevent someone else from doing a similar systematic review. I’m also not convinced that it’s necessary to prevent other investigators from doing a similar study. There have been multiple systematic reviews on treatment in Duchenne before this one after all.

Article summary: The 5th bullet point still mentions the use of nutraceuticals, although the authors reportedly removed this from the search.

Introduction, State of the art: Please use “Becker muscular dystrophy” here. It is not called “muscular dystrophy of Becker.”

Introduction, State of the art: “The ataluren, which was developed with this same mechanism of action, have demonstrated to be more effective than gentamicin.” Whether ataluren is truly effective in treating Duchenne muscular dystrophy is the topic of some BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from controversy. It might be more accurate to say that ataluren was more effective at promoting nonsense readthrough.

Introduction, State of the art: “Furthermore, whether the early beginning of the management of comorbidities improves the functional status and the prognosis of the patients, as well as which are the most effective drugs for to treat these complications, is an important issue that should be clarified.” It is not stated how the study will examine or address the topic of early management of comorbidities in Duchenne.

Rather than trying to include the name of every possible treatment in the search terms, I wonder if it might be more inclusive to eliminate the second group of search terms altogether. That is, to search: (‘dystrophy’ OR ‘duchenne’ OR ‘dmd’ OR ‘dystrophinopathy’) AND (‘‘randomized clinical trial’ OR ‘randomized controlled trial’ OR ‘clinical trial’) This approach will probably result in many more references, especially in EMBASE, but it would prevent the investigators from missing relevant trials because the drug wasn’t one of the specific search terms. The additional drugs that I had listed in my original comments could be a small subset of the drugs that have been tested in Duchenne over time, and it would defeat the purpose of a systematic review to exclude drugs because we didn’t happen to think of them before conducting the search. This approach would also have the benefit of identifying any nutraceutical trials that may have been done, thus allowing for their inclusion in the review.

Methodology, Study status: “Subsequently, the search will be updated, and quality assessment and data analysis will be conducted.” It is unclear what “subsequently” refers to. Is there a planned update to the search at a specific point? If so, this should be stated. http://bmjopen.bmj.com/

Discussion: “Along with this test, the results of the meta-analysis for NSAAs and TFTs will reinforce the evidence on the efficacy of the treatments that determine these parameters.” What parameters are the authors referring to here?

Discussion: “Furthermore, regarding the 6MWD, two characteristic stages during the course of DMD have been showed: 1) a 'stable' stage, with results greater than 300-350 meters, where functional on September 23, 2021 by guest. Protected copyright. decline is relatively slow; 2) a 'decline' stage, with results less than 300 meters, where ambulation is lost quickly.” I do not believe that this sentence is an accurate representation of the cited article. The paper by Hamuro et al. actually describes a model that predicts a developmental improvement in the 6MWD for children with Duchenne up until the age of 10 followed by a period of decline.

Limitations: “This review will have as potential limitations publication and information bias.” I wouldn’t consider publication and information bias to be limitations of this particular review. Rather, they are a possible discovery in any systematic review, and the discovery of these biases may be informative. In this section, it might be more appropriate to discuss anticipated problems with the search. For instance, given that previous Cochrane systematic reviews have focused on single drug classes (like corticosteroids), how do the authors plan to consolidate data from many different classes of drugs? BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from

VERSION 2 – AUTHOR RESPONSE

Comments to the Author

Reviewer #1

Comments:

• Article summary: The authors state that the study will, “enhance the transparency of research, reducing publication bias, preventing selective publication and selective reporting of research outcomes.” I would consider rephrasing this. A systematic review doesn’t actively reduce publication bias or prevent selective publication because it only evaluates articles after they have already been published. The methodology that the authors describe can identify publication bias or selectivity in reporting, but it doesn’t correct these problems.

Authors

The reviewer´s comment seems judicious. As suggested, we have rephrased this sentence as follows (Page 2):

“This study presents an explicit and replicable methodology to analyze the effect of novel drug treatments in Duchenne muscular dystrophy on functional, histological and biochemical outcomes in order to provide the clinicians a comprehensive synthesis of the evidence of the current pharmacological strategies for this disease, identifying potential risk of bias.”

• Article summary: “The publication of this protocol for systematic reviews and meta-analysis prevents from unnecessary duplication of research by describing which outcomes and selection criteria of trials are planned to be considered in the systematic review and meta-analysis.” I’m not sure that publishing the protocol will actually prevent someone else from doing a similar systematic review. I’m also not convinced that it’s necessary to prevent other investigators from doing a similar study. There have been multiple systematic reviews on treatment in Duchenne before this one after all. http://bmjopen.bmj.com/

Authors

The reviewer´s comment seems judicious. We have removed this bullet poin to avoid misunderstandings.

• Article summary: The 5th bullet point still mentions the use of nutraceuticals, although the authors reportedly removed this from the search. on September 23, 2021 by guest. Protected copyright.

Authors

We apologize for the mistake. Thus, we have removed nutraceuticals from this bullet point (Page 2):

• Introduction, State of the art: Please use “Becker muscular dystrophy” here. It is not called “muscular dystrophy of Becker.”

Authors

Thank you. Done.

• Introduction, State of the art: “The ataluren, which was developed with this same mechanism of action, have demonstrated to be more effective than gentamicin.” Whether ataluren is truly effective in treating Duchenne muscular dystrophy is the topic of some controversy. It might be more accurate to say that ataluren was more effective at promoting nonsense readthrough. BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from

Authors

Thank you for the reviewer’s suggestion. Thus, we have rephrased the stated sentence as follows (Page 3):

“Ataluren, with the same mechanism of action, has shown more efficacy than gentamicin at promoting nonsense readthrough”

• Introduction, State of the art: “Furthermore, whether the early beginning of the management of comorbidities improves the functional status and the prognosis of the patients, as well as which are the most effective drugs for to treat these complications, is an important issue that should be clarified.” It is not stated how the study will examine or address the topic of early management of comorbidities in Duchenne.

Authors

We really appreciate the reviewer’s thoughtful comment. As suggested, we have rephrased the paragraph as follows (Page 4):

“Despite numerous trials testing new treatments for DMD have recently been conducted, there is a lack of evidence about the effectiveness of these treatments on delaying the progression of the disease, as well as the conditioning factors that could modify this effectiveness, such as the baseline characteristics of patients or their age.”

• Rather than trying to include the name of every possible treatment in the search terms, I wonder if it might be more inclusive to eliminate the second group of search terms altogether. That is, to search: http://bmjopen.bmj.com/ (‘dystrophy’ OR ‘duchenne’ OR ‘dmd’ OR ‘dystrophinopathy’) AND (‘‘randomized clinical trial’ OR ‘randomized controlled trial’ OR ‘clinical trial’)

This approach will probably result in many more references, especially in EMBASE, but it would prevent the investigators from missing relevant trials because the drug wasn’t one of the specific search terms. The additional drugs that I had listed in my original comments could be a small subset of the drugs that have been tested in Duchenne over time, and it would defeat the purpose of a systematic review to exclude drugs because we didn’t happen to think of them before conducting the on September 23, 2021 by guest. Protected copyright. search. This approach would also have the benefit of identifying any nutraceutical trials that may have been done, thus allowing for their inclusion in the review.

Authors

Thank you for the reviewer´s comment. As suggested, we have modified the search strategy (Page 5):

“(‘dystrophy’ OR ‘duchenne’ OR ‘dmd’ OR ‘dystrophinopathy’) AND (‘‘randomized clinical trial’ OR ‘randomized controlled trial’ OR ‘clinical trial’).”

• Methodology, Study status: “Subsequently, the search will be updated, and quality assessment and data analysis will be conducted.” It is unclear what “subsequently” refers to. Is there a planned update to the search at a specific point? If so, this should be stated.

Authors BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from

Thank you for reviewer’s comment. We have rephrased the paragraph as follows (Page 7):

“[…] The design of the final search strategy and data extraction form will be developed based on preliminary findings. Formal data search on databases and study selection will be conducted on July 31, 2019. Then the quality assessment and the final data analysis will be performed.”

• Discussion: “Along with this test, the results of the meta-analysis for NSAAs and TFTs will reinforce the evidence on the efficacy of the treatments that determine these parameters.” What parameters are the authors referring to here?

Authors

Thank you for the reviewer´s comment. We have rephrased the paragraph as follows (Page 7):

“Likewise, in addition to the 6MWD analysis, the results of the meta-analysis for TFT and NSAA could reinforce the evidence on the efficacy of these treatments. Although the change in 6MWD is the main outcome used in clinical trials, changes in TFT and NSAA are associated with the progression of the disease.”

• Discussion: “Furthermore, regarding the 6MWD, two characteristic stages during the course of DMD have been showed: 1) a 'stable' stage, with results greater than 300-350 meters, where functional decline is relatively slow; 2) a 'decline' stage, with results less than 300 meters, where ambulation is lost quickly.” I do not believe that this sentence is an accurate representation of the cited article. The paper by Hamuro et al. actually describes a model that predicts a developmental improvement in the 6MWD for children with Duchenne up until the age of 10 followed by a period of decline.

Authors http://bmjopen.bmj.com/

Thank you for reviewer’s comment that help us to improve the paragraph as follows (Page 7):

“In addition, regarding to 6MWD, two stages are shown in the disease, the first stage with an improvement of 6MWD up to the age of 10 years old, and a second stage with a progressive worsening until the loss of ambulation and wheelchair dependence 25. For this reason, stratification of patients over and under 7 years has been proposed in clinical trials: it is likely that an effective treatment will show greater effect in patients with a greater decline, however, the greatest clinical on September 23, 2021 by guest. Protected copyright. benefit is expected in early stages of the disease27.”

• Limitations: “This review will have as potential limitations publication and information bias.” I wouldn’t consider publication and information bias to be limitations of this particular review. Rather, they are a possible discovery in any systematic review, and the discovery of these biases may be informative. In this section, it might be more appropriate to discuss anticipated problems with the search. For instance, given that previous Cochrane systematic reviews have focused on single drug classes (like corticosteroids), how do the authors plan to consolidate data from many different classes of drugs?

Authors

Thank you for the reviewer´s comment. As suggested, we have rephrased the limitations paragraph including anticipated problems with the search (Page 8): BMJ Open: first published as 10.1136/bmjopen-2019-029341 on 6 September 2019. Downloaded from

“This review will have potential limitations such as those related with the literature search strategy, since some potentially relevant documents (PhD dissertations, abstract of symposiums, etc.) might be missed undermine the sensitivity of the search strategy. Additionally, main multidisciplinary databases (proposed by the Cochrane Handbook for Systematic Reviews of Interventions) will be search but some bias could be produced as the searching of specific databases is not planned.”

Furthermore, the final reviewer´s question seems judicious. As we have described in the study status section, preliminary searches have been conducted to depict the current state of the art in this issue. This fact allows us to plan a standard meta-analysis; however, if we retrieved more studies than expected, we might conduct other methodological approach such as network meta-analysis or umbrella review, so we would upgrade the record in PROSPERO indicating this new methodology.

VERSION 3 - REVIEW

REVIEWER Doris G. Leung Assistant Professor of Neurology Center for Genetic Muscle Disorders Kennedy Krieger Institute USA REVIEW RETURNED 17-Jul-2019

GENERAL COMMENTS My comments and questions for this revision have been adequately addressed.

http://bmjopen.bmj.com/ on September 23, 2021 by guest. Protected copyright.