PONE-D-21-36367Sex-specific and concentration-dependent influence of Cremophor RH 40 on ampicillin absorption via its effect on intestinal membrane transportersPLOS ONE

Dear Dr. Deng,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Feb 05 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Chun-Hua Wang

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at 

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and 

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. 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.

3. Thank you for stating the following in the Acknowledgments Section of your manuscript: 

"We are grateful to Dr. Yang Mai for her advice and guidance on the subject.. We also thank the scientific research platform provided by Sun Yat-Sen University for the smooth progress of our experiments. This work was supported by the financial support from the Shenzhen Science and Technology Program (Grant No. KQTD20190929173853397), and the National Natural Science Foundation of China (Grant No. 81772449 and 81971081)."

We note that you have provided funding information that is not currently declared in your Funding Statement. However, funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form. 

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: 

"Dr. Wenbin Deng received the awards and this work was supported by the financial support from the Shenzhen Science and Technology Program (Grant No. KQTD20190929173853397), and the National Natural Science Foundation of China (Grant No. 81772449 and 81971081).

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

4. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript. 

5. Please include a copy of Table 1 which you refer to in your text on page 10.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

Reviewer #5: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: I Don't Know

Reviewer #5: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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: No

Reviewer #4: Yes

Reviewer #5: Yes

**********

4. 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

Reviewer #4: Yes

Reviewer #5: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The topic is so interesting and the research findings are well presented. Authors had discussed their results appropriately and had selected good references to support it.

The only remark is that the resolution of Figure 1 needs improvement because it is not readable.

Reviewer #2: Line 71-73 Cite a reference number for this statement.

101 Define what is PBS

104-107 Provide the number of male rats and the number of female rats used for the study separately. In the section 2.3.

127 Define what is LHD

143 According to the manuscript pharmacokinetic parameters of Cmax, tmax, AUC0-240 and AUC∞ have been calculated. However, in the results section all these parameters except AUC0-240 have to be deduced by the reader from the Figure 1b. It is required to provide the results for these parameters with the Figure 1b or separately.

145 Describe the positive and negative controls used in determining the effect of cremophor RH 40 on intestinal membrane damage.

164 Remove “a” before at

181 Provide the reference number for MacLean's study here

241-242 It is not clear the shown plasma concentration values are corresponding to what inlet concentration/s of ampicillin. Provide this information both in the paragraph here and in Figure 1b

255-257 Rather than given a range of 0.03% - 0.07%, provide the 3 Cremophor RH 40 concentrations resulting 41%, 32% and 25% increase in ampicillin absorption

310-312 If this hypothesis of possible influence of PepT1 is used to explain ampicillin absorption in male rats, how the same argument to be used to explain same thing in female. Need a discussion with respect to female rats on the same line.

318-321 Solubility measurements done is not mentioned in the methodology and in the results section. Therefore, bringing a new result in the discussion is not acceptable. Include solubility measurement method and results in the relevant sections.

Figures Poor contrasting and clarity. Need improvements

Figure 1 Write the units of the y-axis

Reviewer #3: The authors delivered a considerable work of the sex-specific and concentration-dependent influence of Cremophor RH 40 on ampicillin absorption. This work revealed the mechanisms of Cremophor RH 40 on intestinal membrane transporters to some extent, and supplied some valuable experience to evaluate the use of Cremophor RH 40 in the design and development of oral delivery systems.

However, there are still some aspects to discuss.

1. It seems irrational or inexactly that the dose of Cremophor RH 40 in the experiment was noted as volume in volume, since Cremophor RH 40 as a semi-solid excipient at room temperature, cannot be exactly measured by volume even at heated status. Also the weight dose of ingredients administrated orally was preferred rather than volume.

2. According to the results of AUC and Cmax after oral administration of 50mg/kg ampicillin mentioned in reference 9, the AUC and Cmax herein were 5-fold higher in male rats after 5mg/kg ampicillin injected into jejunum closed-loop (one tenth of the dose in reference 9). Explanations with more details should be available in line 299 of the manuscript.

3. The pharmacokinetic parameters (Cmax, tmax, AUC0-240 and AUC∞) calculated in section 2.4.4, should be listed as a table form in the manuscript.

4. Some spelling mistakes should be well corrected in line 92,164,267, etc.

Reviewer #4: The research presented in the work “Sex-specific and concentration-dependent influence of Cremophor RH 40 on ampicillin absorption via its effect on intestinal membrane transporters” is an interesting topic in relation to the most widely used excipient Cremophor RH 40 which is an important component of most pharmaceutical preparations.

The following are specific comments, questions and suggestions to improve the quality of the manuscript:

1. Since the focus of the manuscript was to find the effect of the widely-used pharmaceutical excipient Cremophor RH 40 on the intestinal absorption of ampicillin in male and female rats. Therefore, the title must express the true content of the paper and include the animal that the sex specific is related to, so I suggest addition the of the word (in rats) at the end of the title.

2. The introduction to the work was presented correctly, the only point is that no information was given regarding the excipient Cremophor RH 40, I suggest to add a small paragraph about this excipient and its physicochemical properties in the introduction part.

3. Why Cremophor RH 40 has been choose and not Cremophor EL as an excipient? did the second excipient also have the same effect on intestinal absorption of ampicillins?

4. The selection of methods is rather broad and cover the design-space well as were the reported research results.

5. the following used abbreviations should be defined:

P6 - L 101: PBS

P7 - L 127: LDH

P11- L 208: HEPES

P 12 - L 221: Tma-DPH

P 16 -L 325: CMC

Reviewer #5: The manuscript titled “Sex-specific and concentration-dependent influence of Cremophor RH 40 on ampicillin absorption via its effect on intestinal membrane transporters” reported the effects of Cremophor RH 40 on PepT1 protein recruitment on the membrane and led to the changes of ampicillin absorption in rats, which is interesting and new. The paper could even increase in quality by performing the following amendments:

1. Please explain the basis of the Cremophor RH 40 dosage used in the article and its relationship with the clinical preparations

2. transport experiments on Caco-2 cells are needed to verify the effects of Cremophor RH 40 on the PepT1 protein recruitment on the cell membrane

3. relevant references are missing in line 366

4. try to calculate/estimate the meaning of the interaction of Cremophor RH 40 and ampicillin observed in your experiments. Does the magnitude of interaction and doses reflect the clinical observations in literature? Or is it x-fold lower

**********

6. 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: Yes: Associate Professor/ Sally A. El-Zahaby, phD

Reviewer #2: Yes: Dr. Dhanusha Thambavita

Reviewer #3: No

Reviewer #4: Yes: Shahla S. Smail

Reviewer #5: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment 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. Registration is free. 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 PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachments

Attachment

Submitted filename: Comments.docx

Sex-specific and concentration-dependent influence of Cremophor RH 40 on ampicillin absorption via its effect on intestinal membrane transporters in rats

PONE-D-21-36367R1

Dear Dr. Deng,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Chun-Hua Wang

Academic Editor

PLOS ONE