Dear Dr Thomas,

Thank you very much for submitting your manuscript "Drug reformulation for a neglected disease.  The NANOHAT project to develop a safer more effective sleeping sickness drug." for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

We recognise that you have as a team performed a thorough analyses of this approach to drug treatment, which has failed. We consider this important for the community to know. PLoS NTD readership will be lost with the majority of the data in this paper. We asked one reviewer versed in drug pharmacokinetics to give us suggestions for improving readership and the other versed in the blood brain barrier.

We ask that you take their hard work to heart and move many of the details to supplemental and make the main data accessible to our readership in a more broad fashion. PLoS is committed to publishing all data whether negative or not. We hope that you take our suggestions seriously.

Forgive us for taking so long, these times of COVID work in mysterious ways.

With all good wished Jayne

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Jayne Raper, PhD

Associate Editor

PLOS Neglected Tropical Diseases

Ana Rodriguez

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

We recognise that you have as a team performed a thorough analyses of this approach to drug treatment, which has failed. We consider this important for the community to know. PLoS NTD readership will be lost with the majority of the data in this paper. We asked one reviewer versed in drug pharmacokinetics to give us suggestions for improving readership and the other versed in the blood brain barrier.

We ask that you take their hard work to heart and move many of the details to supplemental and make the main data accessible to our readership in a more broad fashion. PLoS is committed to publishing all data whether negative or not. We hope that you take our suggestions seriously.

Forgive us for taking so long, these times of COVID work in mysterious ways.

With all good wished Jayne

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The objectives were clearly stated, and study design was clearly stated.

Reviewer #2: (No Response)

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: The analysis is consistent with the plan, and completely presented; perhaps TOO completely. See below.

Reviewer #2: (No Response)

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This is a very (very) thorough article that describes the studies aimed at understanding the effect of formulation on CNS activity for pentamidine. The questions were: (1) does the formulation facilitate transmission to the CNS; (2) does formulation affect toxicity or efficacy of pentamidine (and is the formulation itself toxic)? Further experiments were performed in order to fully characterize the formulations.

The outcome of the experiments was less than successful, given the goal for CNS activity of pentamidine. Nonetheless, there is a lot of very useful information here, and the thoroughness of the work affords a great deal of useful information and rigorous data.

That said, this article is much, much too long. The authors would benefit from more careful selection of what experimental designs, details, and results belong in the main body of the paper, and what could be most usefully put into the supporting information. I believe that if this paper were to be published with this level of depth and detail, it should be in a pharmaceutics journal, rather than PLOS NTDs. To the PLOS NTD readership, I believe that this paper is too unfocused, and that the salient experimental results and interpretation should be severely streamlined.

Some specific comments:

1. There are a good number of grammatical errors; the paper needs a careful re-read.

2. Line 143 - how does the successful use of AmpB micelles indicate that pluronics "are also active agents with key biological functions"?

3. Line 151. I think that if there's eivdence that pluronics improve BBB penetration it would be good to reference that here.

4. The Figure 1 content is very dense, the fonts are very small, and the color coding is not explained. In addition, the "SAR feedback loop" is never addressed in the paper -- how would the results feed back into the in silico and subsequent experiments? And I don't believe it's comment for the term "structure activity relationships" to be used referring to formulations...

5. Line 396 and following -- the last sentence of this paragraph is confusing....were only two healthy volunteers' blood used? If so, maybe just say this in the first sentence.

6. Line 471 - what is the difference between "permeability' and "accumulation" formats?

7. In the animal studies, why were opposite sexes used in the two different mouse strains?

8. Table 3 - the last three entries show estimated Ki values. How were these estimated? And all three are estimated to be exactly 10 uM?

9. Table 4. Please consider whether four significant figures are appropriate here.

10. Line 824 and following. I'm surprised to see that pluronics are so active in inhibiting insulin secretion...and/or that this wouldn't already have been a known phenomenon given the formulations' prevelance. IF it is know, this should be stated and referenced.

11. Line 1061. I'm not sure that I saw that any MD simulations were performed, or how they were utilized (they don't seem to be mentioned anywhere else.

Reviewer #2: See attached comments called "Reviewer #2 Comments" or the file named "Reviewer2_PNTD-D-20-00526"

--------------------

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 #2: No

Figure Files:

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

Data Requirements:

Please note that, as a condition of publication, PLOS' data policy requires that you make available all data used to draw the conclusions outlined in your manuscript. Data must be deposited in an appropriate repository, included within the body of the manuscript, or uploaded as supporting information. This includes all numerical values that were used to generate graphs, histograms etc.. For an example see here: http://www.plosbiology.org/article/info%3Adoi%2F10.1371%2Fjournal.pbio.1001908#s5.

Reproducibility:

To enhance the reproducibility of your results, PLOS recommends that you deposit laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see https://journals.plos.org/plosntds/s/submission-guidelines#loc-methods

Attachments

Attachment

Submitted filename: Reviewer2_PNTD-D-20-00526.docx

Dear Dr Thomas,

We are pleased to inform you that your manuscript 'Drug reformulation for a neglected disease.  The NANOHAT project to develop a safer more effective sleeping sickness drug.' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Ana Rodriguez

Deputy Editor

PLOS Neglected Tropical Diseases

Ana Rodriguez

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #2: See comment to Editor

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #2: See comment to Editor

**********

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #2: See comment to Editor

**********

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #2: See comment to Editor

**********

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #2: See comment to Editor

**********

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 #2: No