PONE-D-23-22898Rationale and protocol for a safety, tolerability and feasibility randomized, parallel group, double-blind, placebo-controlled, pilot study of a novel ketone ester targeting frailty via immunometabolic geroscience mechanisms.PLOS ONE

Dear Dr. Stubbs,

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Additional Editor Comments:

The authors need to ensure that the research is properly verified and need to improve the quality of the manuscript. The authors need to hone in on the key points raised by the three reveiwers and correct inadvertent errors in the revised manuscript.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The primary objective of this randomized, placebo-controlled, double-blind, parallel group, pilot trial is to determine tolerability of 12-weeks of KE ingestion in a generalizable population of older adults (≥ 65 years). Secondary outcomes include safety and acute blood ketone kinetics. Exploratory outcomes include physical function, cognitive function, quality of life, aging biomarkers, and inflammatory measures. Overall, this study will evaluate feasibility, tolerability, and safety of KE consumption in older adults and provide exploratory data across a range of geroscience-related endpoints.

All key components of the trial protocol were discussed well, including the experimental design, primary objective, primary endpoint, secondary endpoints, exploratory endpoints, randomization procedure, blinding, sample size estimation, and statistical analysis plan. There are only several minor trial design and statistical concerns.

Statistical critiques:

1. It is unclear why the authors chose the proportion of subjects (binary outcome) reporting the same moderate to severe symptom (among dizziness, headache, or nausea) occurring on more than one day within any given two-week recall period (after week 0–2 acclimation period) to determine the study power instead of the beverage tolerability questionnaire (BTQ) score (continuous outcome).

2. It is unclear why the authors proposed having an independent study medical officer to review the safety lab results and approve continuation of subjects in the study instead of forming a DSMB.

3. Please clearly define the “primary outcome” in the Sample Size section. In addition, the authors should focus on a precision analysis instead of a formal power analysis for their pilot study with only 30 participants.

4. The authors proposed using block allocation sequence for the randomization process. Please clearly specify the block size.

5. In addition to the formal hypothesis test, the authors should focus on the estimation of the study outcomes in the Statistical Analysis section because this is a pilot study.

6. Please clearly specify the statistical methods that will be used for generating the 95% confidence intervals for all the study outcomes.

7. The current length of the paper seems to be too long. The authors may need to move some content to the Supplementary section.

Reviewer #2: 1. Do not use 'parallel group'

2. is this pilot?

3. Do not mention pilot

4. Headline 'Study design' is okay

5. Overall okay.

Reviewer #3: This is a timely manuscript by Stubbs and Newman and colleagues that provides an overview of a randomized, placebo-controlled, clinical trial to examine the effects of a ketone ester on age-related health outcomes in older adults. There is expanding interest in geroscience, but several hurried/poorly designed studies that over-interpret data (e.g., from non-placebo-controlled studies that were primarily planned for safety) are being conducted and/or recently published. Here, the authors provide the rationale and a thoughtful and well-written description of their planned RCT.

Strengths include the study rationale, emphasis on tolerability and safety for a short-term study, and, importantly, an assessment of blood ketone concentrations/kinetics. The collection of outcomes across multiple physiological systems a-la-geroscience is also viewed favorably, although, as the authors note, they are not likely to change in this short-term study.

A few minor comments for the authors to consider:

1. A respectful suggestion: To improve readability and expand readership, the background of the abstract is a dense on lingo that will be foreign to much of the readership (e.g., geroscience interventions, inflammo-metabolic aging mechanisms, geroscience mechanisms). The introduction is outstanding, and simple phrases used there would better set the stage in the abstract. Simpler is better.

2. Many discourage descriptions such as “generalizable” or “representative” to describe clinical trial participants. Often notable differences between those that pursue research study participation and those that do not.

3. A better description of randomization would be valuable. Are their blocks planned to balance groups on BMI, comorbid conditions, or other parameters? This may ultimately enhance interpretation of data.

4. The rationale for n = 30 isn’t clear, “Based on studies of symptoms in older adults (43, 44), we predict the primary outcome rate in the placebo group will be approximately 10%. N = 30 (15 per arm) provides 36% power to detect a 25% increase (from 10% to 35%) in the proportion of subjects meeting this primary outcome in the KE condition with two-sided α=0.10.” This would seem to relate to tolerability, so perhaps better to spell out. Moreover, not sure why 36% power is stated favorably. If just a practical sample size, better to state that up front and then provide the power this provides.

5. Just a comment-- it would be ideal to capture muscle performance and physical function measures on two occasions at baseline to account for learning effects. The placebo arm diminishes this concern, but this is somewhat standard of practice.

6. Defer to the editors, but for transparency, it seems the conflicts of Stubbs and Newman should be noted in the main text.

7. Figure 2 is not legible…at all. Fully appreciate this a conversion issue, but it should be reviewed.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Rationale and protocol for a safety, tolerability and feasibility randomized, parallel group, double-blind, placebo-controlled, pilot study of a novel ketone ester targeting frailty via immunometabolic geroscience mechanisms.

PONE-D-23-22898R1

Dear Dr. Stubbs,

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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Kind regards,

Ian James Martins, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript provide a valid rationale for the proposed study, with clearly identified and justified research questions?

The research question outlined is expected to address a valid academic problem or topic and contribute to the base of knowledge in the field.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

2. Is the protocol technically sound and planned in a manner that will lead to a meaningful outcome and allow testing the stated hypotheses?

The manuscript should describe the methods in sufficient detail to prevent undisclosed flexibility in the experimental procedure or analysis pipeline, including sufficient outcome-neutral conditions (e.g. necessary controls, absence of floor or ceiling effects) to test the proposed hypotheses and a statistical power analysis where applicable. As there may be aspects of the methodology and analysis which can only be refined once the work is undertaken, authors should outline potential assumptions and explicitly describe what aspects of the proposed analyses, if any, are exploratory.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Is the methodology feasible and described in sufficient detail to allow the work to be replicable?

Descriptions of methods and materials in the protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample size calculations, and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Have the authors described where all data underlying the findings will be made available when the study is complete?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception, at the time of publication. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above and, if applicable, provide comments about issues authors must address before this protocol can be accepted for publication. You may also include additional comments for the author, including concerns about research or publication ethics.

You may also provide optional suggestions and comments to authors that they might find helpful in planning their study.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors have responded well to the statistical issues raised in the previous review. There is no further statistical concern about this revised manuscript.

Reviewer #3: Thoughtful response to the review and appropriate edits made to the revised manuscript. No additional concerns.

Reviewer #4: #1 The authors mention uncontrolled liver and kidney disease as exclusionary diagnoses, but please describe the specific criteria.

#2 Regarding allocation, are there any allocation criteria?

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

Reviewer #4: No

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