PONE-D-20-20338

In-vivo assessment of a delayed-release clinical formulation of larazotide acetate indicated

for celiac disease using a novel porcine model

PLOS ONE

Dear Dr. Messenger,

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.

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We look forward to receiving your revised manuscript.

Kind regards,

Vivek Gupta

Academic Editor

PLOS ONE

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2. Thank you for stating the following in the Financial Disclosure section:

"The study was funded by Innovate Biopharmaceuticals (Jay Madan), which is now 9meters (https://9meters.com/). KMM and AB received the award (TSA #91690). Yes, the funder played a role in the study: Study design,review of the data and the manuscript. "

We note that one or more of the authors have an affiliation to the commercial funders of this research study : Innovate Biopharmaceuticals.

2.1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

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We will update your Data Availability statement on your behalf to reflect the information you provide.

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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: No

Reviewer #2: Partly

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

Reviewer #1: No

Reviewer #2: I Don't Know

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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: No

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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: No

Reviewer #2: No

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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 manuscript is not very well articulated. Needs extensive English language revision and making sentences meaningful, short, and formal. Lacks scientific correlation and does not follow or state the regulatory guidelines followed throughout the study. The title does not correlate to the study performed and reported. No proper references are cited. The aims and objectives are poorly defined. A major revision is required and cannot be accepted in the current state. The following are the recommendation the authors need to address and make suitable changes.

1. The abstract seems too descriptive; the authors are advised to make the abstract short and concise.

2. The term “novel’ for the porcine model used is ambiguous, the literature review does show other porcine models for this type of study. The authors need to show ceratin validation of this “novel” method developed or used in this study. This is “novel” accounted if the authors have independently published this porcine protocol and got approval from regulatory agencies or cited by fellow researchers after using this reported method. A clear justification is requested. If the authors want to report only the novel porcine model and analytical method developed then the title has to be changed suitably.

3. The authors are advised not to define what methodologies were followed or developed in the abstract making it verbose, instead, those can be reported as results are highly recommended. Mere mentioning about first development or use of any method in abstract does not increase the scientific quality or acceptance.

4. Including abstract the entire manuscript is recommended for extensive English grammar revision, making sentences formal and direct and not either way.

5. From the abstract, the readers would get confused, whether the author's would like to elevate the porcine model or absorbance of delayed LA formulation in s.intestine- contradicts with aim defined in the abstract. Kindly rephrase.

6. The conclusion in the abstract is poorly defined. Kindly rewrite.

7. Keywords need to be rechecked.

8. The sentence LA is being studied in phase 3 clinical trials using a clinical formulation designed to reach the target site for treatment of CeD in the small intestine [1]”- the reference cited seems no connection to this sentence. Kindly recheck and cite appropriately.

9. The introduction is poorly constructed. The objective defined at the end again contradicts with title and abstract written. How can we use a non-validated animal model for IVIVC? As per which guidelines the authors have performed the comparison? Kindly cite or remove the phrase. Complete revision is highly recommended.

10. The surgical procedures lack citing the references from where at least the method was inspired or developed.

11. The drug formulation lacks to disclose the type of polymers or excipients used to delay or make a trigger release in the duodenum. In-vitro dissolution procedure is not reported or cited. The authors need to carefully address this lacuna.

12. Authors have failed to mention or cite using or as per which regulatory guidelines the analytical method was developed and validated. If validated why all parameters were disclosed.

13. Apart from indicating concentration at distal or proximal duodenum by using analytical method, there is no substantial evidence provided by authors on explaining why the formulation could have restricted and had triggered at a specific site. The authors could have mentioned the polymer or excipient used for mucoadhesiveness, formulate into tablet and take x-ray images to locate and see the presence of the beads and then quantify the LA. This seems to be a more justified approach. The authors need to clarify on this.

14. There are various contamination and infections borne while using probes. The authors did not mention how they have taken measures to minimize this. Authors need to address this.

15. The study has only two studies- one analytical method to quantify LA and porcine model to collect sampling. The authors have failed to constructively frame the IVIVC. The authors re advised to follow regulatory guidelines while drawing a comparison study.

Reviewer #2: In this manuscript, the authors report an in vivo procedure for real-time sampling of intestinal fluid from pigs to profile the concentration of larazotide acetate (LA) for the first time. They developed a sophisticated and sensitive UPLC method for LA quantification at biorelevant concentrations. The in vivo procedure and analytical methods are well-described.

While this in vivo sampling technique holds multiple advantages, the authors need to better elaborate on the correlation of their findings with conventionally used in vitro dissolution studies. This study would be a good addition to the existing literature, following few major revisions as listed below:

Introduction:

1. Along with the publications [14-17] already cited by the authors, it may be worthwhile to include another reference utilizing a very similar in vivo study for intestinal tract measurements in pigs for a different orally administered drug:

Yan, L., Xie, S., Chen, D. et al. Pharmacokinetic and pharmacodynamic modeling of cyadox against Clostridium perfringens in swine. Sci Rep 7, 4064 (2017). https://doi.org/10.1038/s41598-017-03970-9

2. While the introduction provides a good overview of the need for a minimally invasive, real-time sampling method for in vivo GI profiling of an oral drug, it does not provide a concrete conclusion about the stated hypothesis. It will be helpful to add 2-3 sentences describing how (qualitatively and/or quantitatively) the in vitro-in vivo data correlated based on the demonstrated results.

Materials and methods:

1. Line 125: butterfly ‘valve (is missing)’

2. Line 153-154: For reproducibility, it will be helpful to mention the value of consistent ratio maintained for the intestinal fluid and quench solution

3. System suitability:

- Need to rephrase this section as the sentences are confusing due to repeated occurrences of the phrase ‘system suitability’.

- For complete definition of method, please include the standard concentration that was selected for these studies

-What quantitative criteria were set to determine the system suitability/ repeatability?

Results:

1. Reorganize Table 1 to include the volume of intestinal fluid collected from duodenum and jejunum at different time points, respectively and the corresponding LA concentrations

2. Table 1/Fig. 9: It is unclear how n=3 is obtained, if only 2 pigs were used for the study. Are these biological replicates or technical replicates?

Discussion and conclusions:

1. The authors need to revise the discussion sections to improve the flow of concepts and connectivity.

For e.g. The statement in line 262 ‘It has been reported…’ fits better in lines 310/311

2. Line 260: The use of adjective ‘meaningful’ is very vague. It is unclear if they want to imply bio-relevance or agreement with qualitative in vivo release profile, etc.

3. While the authors repeatedly state that the in vivo concentrations correlate well with the in vitro dissolution results, there is no explicit description on how these in vitro results were captured, interpreted and (qualitatively) compared/correlated. It will be helpful to provide a detailed discussion on Fig 3 and its implications for Fig 9.

Figures:

Figure 1 and 2 can be clubbed together

Figure 3:

- Is the in vitro dissolution data published in previous literature, or generated for this study? If reusing from previous literature, please cite the source. If the study was performed for this manuscript, include the methodology and result discussion for the same.

- Edit axes titles to be more descriptive of the units shown: e.g. X axis- time (minutes), Y-axis- cumulative % dissolved

- It is unclear how samples 1-6 are related to each other. Are they replicates? Please edit the legend appropriately.

Figure 5: Elaborate this caption to describe the model and provide legend for notations used (L, S, k, 1,2 etc.)

Figure 6: can be clubbed with Fig. 5 or moved to supplementary information

Figure 7: Multiple spelling errors

Figure 8: can be moved to supplementary information

Figure 9:

- Edit axes titles to be more descriptive of the units shown: e.g. X axis- time (hr), Y-axis- LA concentration in intestinal fluid (μM)

- (Optional) Can also include a similar dotted line to identify LOD

Supplementary information:

(Optional) Include calibration curves and method suitability study data

Copyediting recommendations:

- Need to standardize (italicize and/or remove hyphen) the format of words like ‘in vitro’ and ‘in vivo’, ‘et al’ throughout the document.

- Avoid unnecessary capitalization (e.g. line 135: In-vitro, line 461: Pig)

- Maintain a single space between values and their unit of measurements for standardization (e.g. line 29: ‘1 mg’ instead of ‘1mg’) throughout the manuscript

- Line 91: Capitalization of the letter ‘I’ in the word- institutional

- Line 215: Explicitly define ‘CV’ (as coefficient of variation?)

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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: No

Reviewer #2: 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.

PONE-D-20-20338R1

In vivo assessment of a delayed release formulation of larazotide acetate indicated for celiac disease using a porcine model

PLOS ONE

Dear Dr. Messenger,

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 27 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Vivek Gupta

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. 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: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

**********

4. 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

**********

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

**********

6. 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 following recent works should be cited and discussed in the results & discussion, this would elevate the importance of the study.

Expert opinion on drug discovery 14, no. 10 (2019): 957-968.

Pharmaceutics 12, no. 11 (2020): 1124.

Gut and liver 9, no. 1 (2015): 28.

Pharmaceutics 12, no. 7 (2020): 652.

Expert Opinion on Pharmacotherapy 12, no. 11 (2011): 1731-1744.

Processes 8, no. 3 (2020): 316.

Gastroenterology 148, no. 7 (2015): 1311-1319.

Peptides 35, no. 1 (2012): 86-94.

Reviewer #2: (No Response)

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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 #2: 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.

In vivo assessment of a delayed release formulation of larazotide acetate indicated for celiac disease using a porcine model

PONE-D-20-20338R2

Dear Dr. Messenger,

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,

Vivek Gupta

Academic Editor

PLOS ONE