PONE-D-19-17798

Gravity influences bevacizumab distribution in an undisturbed balanced salt solution in vitro

PLOS ONE

Dear A/porf. Ra,

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2. Please include the underlying raw data in a Supporting Information file, namely the individual concentration measurements for each tube at each timepoint.

Additional Editor Comments:

Please address concerns of reviewers 1 and 2 carefully especially the concern about using test tubes and how clinically applicable your study is.

Please also temper your conclusions based on the concerns of the reviewers.

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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: Yes

Reviewer #2: No

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: I Don't Know

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

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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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 findings of the authors are interesting. However, based on the data presented, there seems to be no way to apply these purely in vitro findings to clinical practice at this time. Therefore, the concluding sentence of results is an overstatement and not supported by the results presented, “These factors should be taken into account during intraocular bevacizumab injection after vitrectomy.” Recommend this be eliminated or modified substantially to directly and accurately reflect the results presented.

Similarly for the comments made in lines 137-138: there is no data in the current manuscript to support the claim that “similar results would be observed in clinical practice.” This should be removed or substantively modified. Also for lines 153-154, there is no data to indicate that the current results “need to be taken into account during intraocular injection.” Again, over reach by the authors beyond a reflection of their actual, presented data. It would be acceptable to mention these issues as possibilities requiring more study, particularly in vivo studies. But, to conclude that human treatment should be directly modified based on this work alone should be avoided.

The authors state that a “micropipette” was used to collect the samples from the tubes at the end of the prespecified waiting period. How was this performed for the “lower layer” without disturbing the “upper layer” in the process. Or, was all of the upper layer removed and then the lower layer was accessed? Please clarify exactly how the samples were harvested prior to ELISA analysis. This seems to be partially addressed in lines 163-166, but clarity in the Methods would be valuable to readers.

Reviewer #2: The study aims to evaluate the effect of gravity on bevacizumab in vitro.The authors added bevacizumab to the upper part of a test tube filled with balanced salt solution (BSS) and examined its distribution over time. They found significant differences in concentration between the upper and lower parts of the test tubes, even after a considerable amount of time had passed which shows that bevacizumab did not dissolve immediately and diffuse evenly throughout the solution. They reasoned that more bevacizumab settles in the lower part of the tube relative to the upper part because of gravitational force.

The biggest problem of this study is they used test tubes to conduct the study which is so different from the actual environment in the eye. This will make the data difficult to be interpreted in the "in vivo" condition.

Other comments:

1. Please describe the material of test tubes, which will be a possible factor to affect the distribution of medications

2. Please describe the meaning of all the abbreviations

3. In discussion, authors stated that bevacizumab was injected at a constant injection speed, however, in material and methods parts, the way of injection was not described, which would also cause the difference in distribution. Please also state further how to keep a constant injection speed.

4. The method of temperature control should be mentioned

5. In the first paragraph of discussion, authors stated that the concentration was higher in the upper part after 6 to 168 hours after injection, however, the first measurement time stated in your methods was 12 hours after the injection.

Minor points:

6. Line 56: "bevacizumab injection is often prescribed after vitrectomy", may change to "Bevacizumab injection is often given, or performed, after vitrectomy

7. Line 60: “whether bevacizumab dissolves and disperses rapidly and evenly throughout the vitreous chamber when injected into balanced salt solution (BSS) filling the vitreous chamber after vitrectomy”, may change to “when injected into balanced salt solution (BSS) filled eyes”

8. Line 65: “Through this, we aimed…”, you can delete the word “Through this” and start the paragraph with “We aimed…”

9. Line 90: “Bevacizumab concentrations...” should be written as “Bevacizumab’s concentrations...”

Reviewer #3: I believe this research may have other applications besides DR. For instance, Rop and Srn.

Can the authors please comment on thoughts on the same concept with an intact vitreous?

It would be interesting to do an animal model for the distribution of antivegf in eyes with intact vitreous.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Clio Armitage Harper III

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Gravity influences bevacizumab distribution in an undisturbed balanced salt solution in vitro

PONE-D-19-17798R1

Dear Dr. Ra,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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With kind regards,

M. Elizabeth Hartnett

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments: