Brimonidine and timolol concentrations in the human vitreous and aqueous humors after topical instillation of a 0.1% brimonidine tartrate and 0.5% timolol fixed-combination ophthalmic solution: An interventional study

Yusuke Orii; Eriko Kunikane; Yutaka Yamada; Masakazu Morioka; Kentaro Iwasaki; Shogo Arimura; Akemi Mizuno; Masaru Inatani

doi:10.1371/journal.pone.0277313

Peer Review History

Original SubmissionJune 12, 2022
21 Aug 2022 Decision Letter - Callam Davidson, Editor PONE-D-22-13271Brimonidine and timolol concentrations in the human vitreous and aqueous humors after topical instillation of a 0.1% brimonidine tartrate and 0.5% timolol fixed-combination ophthalmic solution: An interventional studyPLOS ONE Dear Dr. Inatani, 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 Oct 03 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 editorial office and reviewers. 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, Callam Davidson Editorial Office 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 Authors EK and AM are affiliated with Senju Pharmaceutical Co., Ltd. This must be declared as a conflict of interest (via the Submission Form). Additional Editor Comments (if provided): PLOS ONE requires that studies be conducted rigorously. Sample sizes must be large enough to produce robust results, where applicable. Experts in the field have reviewed the manuscript and have noted concerns regarding your sample size calculation. Please ensure you address these comments in your rebuttal letter and revised manuscript. Reviewer #3 suggest the inclusion of a control group, however given that your study was associated with a prospective protocol and inclusion of a control group at this time may prove difficult, an alternative would be to discuss the absence of a control group as a limitation in your Discussion. Any changes to the protocol-specified design or analyses ought to be described as such in the Methods. [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: Partly Reviewer #3: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No Reviewer #3: No ****** 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 Reviewer #3: 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 ****** 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: It is unclear how they calculated sample size for this intervention study. What are the type 1 and 2 error, mean, standard deviation that were used for sample size calculation? Small sample size of this study is one of the most significant drawbacks that precludes the results convincing. Furthermore, comparison of drug concentration between phakic and pseudophakic eyes were not performed due to small sample size. When and how the IOP was measured before and after surgery? Did they consider diurnal variation of IOP? Did they measure IOP with Goldmann applanation tonometry? Please add best corrected visual acuity and cirumpapillary retinal nerve fiber layer thickness and or macular ganglion cell/inner plexiform layer thickness before and after surgery. Reviewer #2: The authors reported about a single arm open interventional trial to investigate the concentrations of Brimonidine and timolol in the human vitreous and aqueous humors after topical instillation. They concluded that the majority of patients who received a 0.1% brimonidine tartrate and 0.5% timolol topical fixed-combination ophthalmic solution showed a brimonidine concentration >2 nM in the vitreous humor. The present explorative trial needs some minor modifications. Let me give some detailed comments: 1. The authors are welcome to add individual patient data in a file to facilitate the scientific review process. Note, that the data may be anonymized at this stage. 2. The primary endpoint is not stated. If the primary endpoint variable is "brimonidine concentration >2 nM" the analysis should result in a rate followed by a 95% confidence interval. 3. The presented correlation coefficient should be followed by a 95% confidence interval instead of a statistical test. 4. Please check for decimal point and comma (see line 35). 5. Before after comparison by t-Test (see L141) is difficult to interpret because of regression to the mean and should be avoided. Please use difference of mean with sd and 95% CI for interpretation (see L164). Reviewer #3: Dear authors, This manuscript was reviewed properly. This is a small study aiming to determinate the concentration of timolol and brimonidine in the human vitreous body. Although, the design is sound, there are flaws as follows: 1. There are doubts related to sampling: a. Due to the very spread results of both drug concentrations (mainly in the vitreous body), a control group may help compare very low values. b. Besides, sample size calculation will also be necessary even considering the explanations made by the authors in the text. 2. A comparison with results from patients treated with non-fixed combination of timolol and brimonidine will be of benefit. 3. Although species and method differences are clear from the Suzuki's and cols. study (reference #29), authors should discuss better the why their study deserves additional publication. ****** 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 Reviewer #3: 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. https://doi.org/10.1371/journal.pone.0277313.r001
Revision 1
29 Sep 2022 Author Response Dear Editors and Reviewers. I am sending with our fully revised manuscript entitled “Brimonidine and timolol concentrations in the human vitreous and aqueous humors after topical instillation of a 0.1% brimonidine tartrate and 0.5% timolol fixed-combination ophthalmic solution: An interventional study” (Manuscript ID: PONE-D-22-13271), which I would like to submit for publication in PLOS ONE. We have addressed the point raised by referee as much as possible. The comment from reviewer was very valuable and essential to make our manuscript substantially better. We think now that our revised manuscript is worthy to be considered for publication in the great journal, PLOS ONE. Replies for reviewer’s comments are as follows. Reviewer #1 It is unclear how they calculated sample size for this intervention study. What are the type 1 and 2 error, mean, standard deviation that were used for sample size calculation? Small sample size of this study is one of the most significant drawbacks that precludes the results convincing. Furthermore, comparison of drug concentration between phakic and pseudophakic eyes were not performed due to small sample size. In the phase 1 trial of Aibeta®, each sample size was between 7 and 9 to evaluate for its safety and pharmacokinetics. We followed the sample size in this study. We added the following sentences in the Materials and Method section, “Also, in the phase 1 trial of Aibeta®, each sample size was between 7 and 9 to evaluate for its safety and pharmacokinetics” (p.7, line 109-110 in the revised version). As for the comparison of drug concentrations between phakic and pseudophakic eyes, we had examined drug concentrations in phakic eyes as well as pseudophakic eyes ([17] Takamura Y., Tomomatsu T., Matsumura T., “Vitreous and aqueous concentrations of brimonidine following topical application of brimonidine tartrate 0.1% ophthalmic solution in humans.,” J Ocul Pharmacol Ther., 31(5):282-5, 2015.). However, there was no significant difference of the brimonidine concentrations in the vitreous between the 2 groups. We didn’t recruit more numbers of pseudophakic patients to examine whether the vitreous in the pseudophakic eyes contained more concentration of brimonidine. Actually, the concentration of brimonidine in the pseudophakic eye was 3.77 nmol, which was still lower than the mean brimonidine concentration of all the eyes. When and how the IOP was measured before and after surgery? Did they consider diurnal variation of IOP? Did they measure IOP with Goldmann applanation tonometry? Because the main purpose of the present study was to evaluate for the drug concentrations of the vitreous, we did not consider diurnal variation of IOPs to reduce the burden on the recruited patients. Therefore, IOPs were measured without considering the diurnal change in the time-points before the start of the administration as well as before the surgery. We did not use the Goldmann applanation tonometry because of the patients with macular hole or epiretinal membrane. IOPs were measured with non-contact tonometer to avoid patient distress. We described the following sentence in the method. “Because patients scheduled for pars plana vitrectomy to treat macular holes or idiopathic epiretinal membranes were not associated with glaucoma, the non-contact tonometer (Nidek, Nagoya, Japan) was used to measure IOPs in order to reduce patient distress.” (p.9, line 140-142 in the revised version). Please add best corrected visual acuity and cirumpapillary retinal nerve fiber layer thickness and or macular ganglion cell/inner plexiform layer thickness before and after surgery. As suggested by the reviewer #1, we added the data for best corrected visual acuity at the following sentences in the revised manuscript. Because of the patients with macular hole or epiretinal membrane, we did not evaluate the cpRNFLT or macular ganglion cell complex to avoid excessive testing. Especially, the thickness of macular ganglion cell complex would not provide reliable data in patients with macular hole or epiretinal membrane. In the Secondary outcome measures section: “and best-corrected visual acuity (BCVA) before and after surgery.” (p.9, line139-140 in the revised version) In the Result section: “Mean BCVA in logMAR before and 1 month after surgery were 0.31 ± 0.15 and 0.19 ± 0.09. However, there was no significant difference of the BCVA between the two visits (p = 0.0716). ” (p.11, line 180 - 181 in the revised version) Reviewer #2 The authors are welcome to add individual patient data in a file to facilitate the scientific review process. Note, that the data may be anonymized at this stage. We have added the individual data in a file and upload it. The primary endpoint is not stated. If the primary endpoint variable is "brimonidine concentration >2 nM" the analysis should result in a rate followed by a 95% confidence interval. As reviewer points out, the primary endpoint is to determine whether the brimonidine concentration in the vitreous is enough (>2 nM). We calculated the 95% CI of the brimonidine concentration in vitreous and the result was 1.62-8.45. It spans across 2 nM, but the mean concentration of brimonidine was 5.04 ± 4.08 nM, which is more than 2 nM. This value is almost the same as the result of our previous study with Aiphagan® and majority of patients (63%) showed more than 2 nM of brimonidine in vitreous. We added the 95% CI of the brimonidine to the result section: “The mean brimonidine concentrations in the vitreous and aqueous humors were 5.04 ± 4.08 nM (95% CI: 1.62 - 8.45)” (p.10, line 162 - 163 in the revised version) The presented correlation coefficient should be followed by a 95% confidence interval instead of a statistical test. We calculated the 95% CI of coefficient of determination and added the result in revised manuscript: “95% CI of coefficient of determination were 0.903-0.997 in vitreous and 0.880-0.996 in aqueous humor.” (p.10, line 172 - 173 in revised version) Please check for decimal point and comma (see line 35). Thank you for your suggestion. Decimal point and comma are appropriately arranged. Before after comparison by t-Test (see L141) is difficult to interpret because of regression to the mean and should be avoided. Please use difference of mean with sd and 95% CI for interpretation (see L164). We calculated the difference of mean IOP between before and after instillation and 95% CI of the IOP difference. The following sentence is added in the revised manuscript. In Results session: “demonstrating a significant difference (p = 0.0094; -2.56 ± 0.72 mmHg; 95% CI: -0.85 to -4.27) in IOP before and after administration.” (p.10, line 175 – 176 in the revised manuscript) Reviewer #3 1. There are doubts related to sampling: a. Due to the very spread results of both drug concentrations (mainly in the vitreous body), a control group may help compare very low values. In our previous study ([17] Takamura Y., Tomomatsu T., Matsumura T., “Vitreous and aqueous concentrations of brimonidine following topical application of brimonidine tartrate 0.1% ophthalmic solution in humans.,” J Ocul Pharmacol Ther., 31(5):282-5, 2015.), we have already checked the drug concentration in vitreous of control patients who received no drug instillation and determined values below the lower limits of quantitation. To recruit patients during the registration period facing COVID-19 pandemic world-wide, we skipped the negative control group in this present study. We added the following sentences in the revised manuscript. In Materials and Methods section: “In our previous study, we have already checked the drug concentration in vitreous of negative control patients who received no drug instillation and determined values below the lower limits of quantitation.” (p.8, line 126-128 in revised manuscript) b. Besides, sample size calculation will also be necessary even considering the explanations made by the authors in the text. As recommended by the reviewer #3, we added the explanations about the sample size in the revised manuscript. ” Also, in the phase 1 trial of Aibeta®, each sample size was between 7 and 9 to evaluate for its safety and pharmacokinetics” (p.7, line 109-110 in the revised version). 2. A comparison with results from patients treated with non-fixed combination of timolol and brimonidine will be of benefit. As reviewer #3, the data from patients treated with non-fixed combination of timolol and brimonidine would be better. However, we need to minimize the sample size to avoid the risks for adverse effects because the recruited patients were patients with macular hole or epiretinal membrane who had scheduled vitrectomy. Brimonidine frequently causes allergic conjunctivitis and timolol causes bradycardia and worsens asthma and COPD. We did not measure the concentrations of brimonidine and timolol after the instillation of non-fixed combination so that the CRB committee approved the clinical study. 3. Although species and method differences are clear from the Suzuki's and cols. study (reference #29), authors should discuss better the why their study deserves additional publication. In animal eyes, the penetrance of the eye drops may be different from human eyes. Therefore, we need to confirm the pharmacokinetics about the brimonidine concentration in the human vitreous. This study is the first to evaluate brimonidine concentrations in the vitreous humor after topical instillation of a 0.1% brimonidine tartrate and 0.5% timolol fixed-combination ophthalmic solution in human eyes. We added the description in the discussion: “To the best of our knowledge, this study is the first to evaluate brimonidine concentrations in the vitreous humor after topical instillation of a 0.1% brimonidine tartrate and 0.5% timolol fixed-combination ophthalmic solution in human eyes.” (p.12, line 210-212 in revised version) Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0277313.r002
25 Oct 2022 Decision Letter - Daisuke Nagasato, Editor Brimonidine and timolol concentrations in the human vitreous and aqueous humors after topical instillation of a 0.1% brimonidine tartrate and 0.5% timolol fixed-combination ophthalmic solution: An interventional study PONE-D-22-13271R1 Dear Dr. Inatani, 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, Daisuke Nagasato Academic Editor PLOS ONE Additional Editor Comments (optional): 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: (No Response) Reviewer #2: All comments have been addressed Reviewer #4: (No Response) ******** 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: Yes Reviewer #2: Yes Reviewer #4: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #4: Yes ****** 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: No Reviewer #2: No Reviewer #4: 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 Reviewer #4: 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: (No Response) Reviewer #2: (No Response) Reviewer #4: (No Response) ****** 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 Reviewer #4: No ******** https://doi.org/10.1371/journal.pone.0277313.r003
Formally Accepted
21 Nov 2022 Acceptance Letter - Daisuke Nagasato, Editor PONE-D-22-13271R1 Brimonidine and timolol concentrations in the human vitreous and aqueous humors after topical instillation of a 0.1% brimonidine tartrate and 0.5% timolol fixed-combination ophthalmic solution: An interventional study Dear Dr. Inatani: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. 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. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Daisuke Nagasato Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277313.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .