Dear Dr Chunhui Hu ,

Thank you very much for submitting your manuscript "Improve Anti-alveolar Echinococcosis Efficacy by a Novel Albendazole-Bile acids Derivatives with Enhanced Oral Bioavailability" 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 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,

Alessandra Morassutti, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Jennifer Keiser

Section 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 #1: - Objectives are clearly stated

- The study design is appropriate

- The population is appropriate

- The sample size is adequate to address the hypothesis

- Correct statistical analysis is used

- There are no ethical concerns

Reviewer #2: (No Response)

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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 relates with the study plan

- Results are clearly stated

- The tables and figures are clear

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: - Yes

- No

- This section needs elaboration

- Yes

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: The language has some issues. Appropriate sentence structure is required at many places in the manuscript. The authors should also mention references where required.

Limitations of the study should be stated and there is no comparison of efficacy of this approach with other approaches used for this purpose. Even though, this approach presents a possible route to control the AE, there should be emphasis on its significance and its employability in comparison to the other studies.

Reviewer #2: (No Response)

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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: The study is of significance as it represents one of the novel approaches for the treatment of AE which is affecting a huge chunk of human population. Data are well presented.

Reviewer #2: Alveolar echinococcosis (AE) is an orphan disease as well as a really neglected disease. Anti-AE therapeutic drugs are generally not a focus for pharmaceutical companies due to the economics or the lack of a viable market, and the drug R&D primarily depends on scientific researchers and NPOs. Although several efforts have been made in the last two decades, the anti-AE drug R&D is proceeding slowly; in contrast, the disease burden is still high. So far, albendazole (ABZ) remains the only drug treatment option, but suffers from the drawback of low oral bioavailability. In this context, discovering new chemical entities for the treatment of Echinococcus multilocularis infections should be encouraged. In the present work, the authors designed an albendazole bile acid derivative (ABZ-BA) with the purpose to improve the bioavailability of the parent drug ABZ through changing the crystal form and employing the sodium dependent bile acid transporter expressed in the small intestine. The study is designed reasonably. The chemical synthesis and structural characterization have been done thoroughly, but the work regarding to anti-parasitic activity as well as PK seems insufficient. The manuscript cannot be accepted unless the following major concerns have been addressed.

Major

1. The major drawback of the study is the lack of a therapeutic efficacy study in experimentally infected mice. According to the “Protoscolex collection and cultivation” section, the authors obviously have the access to E. multilocularis infection animal model. In vivo efficacy study must be accomplished before acceptance. It should be verified that whether the increased bioavailability of ABZ-BA claimed by the authors could translate to improved anti-AE treatment outcome.

2. The in vitro anti-parasitic activity of ABZ-BA was only evaluated against protoscoleces but not metacestodes, while the latter stage is the primary parasite lesions in the intermediate hosts (e.g., humans and mice). The anti-parasitic ability of ABZ-BA against metacestode vesicles should be determined and compared with the parent drug ABZ to make sure that the structural derivatization did not exert negative impact on the activity.

3. The authors claimed that “sodium dependent bile acid transporter (ASBT) contributes for the enhanced bioavailability of ABZ-BA through effective transport of bile acids”, but no experimental evidence was provided.

Minor

1. The authors should add a statement why chose 4-amino-1-butanol as a linker? Is there a small-scale screening to identify an appropriate linker? If yes, please provide the screening design and results.

2. How many replicates were carried out to evaluate the protoscolecidal activity of ABZ-BA? In the terms of in vitro bioassay, at least three replicates are required.

3. For PK study, the authors should take into consideration the gender difference, please explain why only male rats were employed in the PK study. Also specify the formulations of ABZ and ABZ-BA for oral administration; I suppose both drugs are not water soluble.

4. Have the authors evaluated the toxicity of the prepared ABZ-BA? Sometimes derivatization could result in elevated risk of toxicity. If not, I suggest cytotoxicity against human liver cells should be examined as least, as AE primarily affects the liver.

5. The pharmacokinetic parameters, T1/2, clearance rate, F%, should be added into Table 5.

6. For Figures 3 and 4, make a clear statement that which kind of statistical analysis was used and add it into the figure legends.

7. For Figures 6, the authors should not include the data of ABZ-BA, because only the plasma concentration of the parent drug ABZ matters for the comparison. The current figure design confuses the readers. Same problem in Figure 3A.

8. Figure 7 is needless, should be incorporated into Figure 1.

9. The authors should provide the product information (cat#, manufacturer, place of production) for all reagents and materials. Some of the information is missing in the Methods section, please check it carefully.

10. The authors should provide the detailed method of isolation of E. multilocularis protoscoleces, since the protocol is complex, and cannot be simplified as the description “Protoscolex (PSC) of E. multilocularis was (not were) collected aseptically from alveolar echinococcosis obtained from the abdominal cavity of gerbils”.

11. The authors could put “Method validation of Ultra High-Performance Liquid Chromatography (UHPLC)-MS” section and its related methods into a supporting information file, which obviously is not the focus of the work.

12. A detailed ethic statement is necessary for the study, and makes it an independent section.

13. The written English of the manuscript must be subjected to a professional language service and requires the editor’s extra attention.

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

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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, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Dear Ph.D Hu,

Thank you very much for submitting your manuscript "Improvement of Antialveolar Echinococcosis Efficacy of  Novel Albendazole-Bile acids Derivatives with Enhanced Oral Bioavailability" 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. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

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

[1] A letter containing a detailed list of your responses to all 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.

Thank you again for your submission to our journal. 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,

Alessandra Morassutti, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Jennifer Keiser

Section 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 #1: (No Response)

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: (No Response)

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: The manuscript has greatly improved in terms of clarity, understanding and English language.

Just a few suggestions are given for correction of the sentences:

L70: Omit ‘of the’

L 75: Echinococcus multilocularis should be in italics

L 85: multilocularis is the name and it should be written in its full form

L 86: humans and animals

L 94-96: reference/s missing

L 97-98: This is repetition of what you have stated in opening paragraph

L 120: Rephrase this sentence for more clarity

L 166: bile instead of bill

L 327: ABZ-BA

Reviewer #2: I am glad to see the authors have made efforts to improve the scientific quality of the manuscript. Before acceptance, there are still a few concerns to be addressed.

1. One of the issues is the newly added “Pharmacodynamic evaluation” section. I suppose all the drug formulations were orally given, as it is the proper way to prove the hypothesis of bioavailability improvement, but the authors gave a statement of administration with “injection”. Please give an explanation.

--How long to initiate the drug administration after infection?

--There is no clear statement of animal housing conditions, animal grouping, and administration period in the method section.

--The description of “pharmacodynamic evaluation” is not a common use, “in vivo therapeutic efficacy in an in-situ AE animal model” would be better.

2. The other concern is the statistical analyses. Comparisons between two groups should use the t-test. For comparisons among multiple groups (≥ 3), one-way ANOVA followed by a reasonable post-hoc test (e.g. Tukey’s) or a nonparametric test (e.g. Kruskal–Wallis) should be used. For example, t-test for the comparison of two groups ABZ and Abz in Fig. 4A, and one-way ANOVA or Kruskal–Wallis test for the three groups in Fig. 7D. The authors should recheck the statistical analysis throughout the manuscript.

3. There is no oral bioavailability F% in Table 1, which is a crucial parameter.

4. The improvement of F% and the reduction of cyst volume should be highlighted in the abstract and author summary.

5. For Fig. 5A, 5D and 5E, replace the legends of Group 1-7 with drug names and concentrations.

6. Change all the mass concentrations to molar concentrations in Figures 4-6 and Table 1, easy to make a comparison of ABZ and ABZ-BA.

7. The language editing requires extra attention of the editors.

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

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, we recommend that you deposit your 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. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

References

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article's retracted status in the References list and also include a citation and full reference for the retraction notice.

Dear Dr Chunhui Hu, We are pleased to inform you that your manuscript 'Improvement of Antialveolar Echinococcosis Efficacy of  Novel Albendazole-Bile acids Derivatives with Enhanced Oral Bioavailability' 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,

Alessandra Morassutti, PhD

Academic Editor

PLOS Neglected Tropical Diseases

Jennifer Keiser

Section 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 #1: (No Response)

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: (No Response)

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: The article is in much better shape and form now but I would again ask to check the English language and make revisions where required.

Reviewer #2: (No Response)

**********

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