PONE-D-20-00899

Pharmacological and pharmacokinetic profile of the novel ocular hypotensive prodrug CKLP1 in Dutch-belted pigmented rabbits

PLOS ONE

Dear Dr Fautsch,

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.

Several concerns, clearly detailed in reviews, should be carefully addressed.

We would appreciate receiving your revised manuscript by Apr 19 2020 11:59PM. When you are 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable 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. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled '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.

We look forward to receiving your revised manuscript.

Kind regards,

Ted S Acott, PhD

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 http://www.plosone.org/attachments/PLOSOne_formatting_sample_main_body.pdf and http://www.plosone.org/attachments/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. To comply with PLOS ONE submissions requirements, please provide methods of sacrifice in the Methods section of your manuscript.

[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

**********

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

Reviewer #1: No

Reviewer #2: 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

**********

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

**********

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: Chowdhury et al present the pharmacological and pharmacokinetic data of a novel prodrug, CKLP1. Overall the study was well designed and demonstrates the necessary profile of this novel drug as a potential therapeutic target. The following suggestions would improve the manuscript:

1. In Figure 2 the authors reference the half-life CKLP1 and Levcromakalim. Can this be highlighted better in the figure? The number is referenced in the text but it would be useful to denote the half-life on the graph as well.

2. Lines 191-192 references Figure 3 and the peak concentration comparisons between days. Can this data be statistically compared?

3. Figure 4, Can this data also be statistically compared (Line 214?

4. Provide more detailed methods on how IOP was measured and what statistical analysis was performed on these data.

Reviewer #2: PONE-D-20-0899

Summary

In this well-written article, the authors study chromakalim prodrug 1(CKLP1) and its derivative, levcromakalim. These drugs can reduce IOP which is important because there is elevated intraocular pressure (IOP) in glaucoma. They focus on the pharamcokinetics of these drugs in Dutch-Belted pigmented rabbits. In this paper, the authors can detect both molecules using LC-MS/MS. In an CKLP1 intravenous delivery model, they measure pharmacokinetic parameters in the blood. The salient findings are that both molecules are detected with levcromakalim coming on slower, suggesting that CKLP1 is converted to it. Then, there are two topical CKLP1 delivery paradigms. The first is short term with both eyes of the rabbits receiving topical drug for 1-, 4-, or 8-days. The second is a longer experiment with one eye receiving topical drug (the other eye served as a vehicle control) for 90-days. Blood, organs, and intraocular contents (aqueous and vitreous) were collected for analyses at various time points. In the,

1-, 4-, and 8-day experiments CKLP1 was not detected in the aqueous or vitreous.

1-, 4-, and 8-day experiments levcromakalim was detected in the aqueous and vitreous.

In the 90-day experiment CKLP1 was not detected in the aqueous or vitreous

In the 90-day experiment levcromakalim was sometimes detected in the aqueous and vitreous.

In the 90-day experiment neither were detected in plasma

Further, the 90-day topical drug study did not lead to observable histological changes to the eye and multiple other organs.

Ultimately, there are some question about the methods and about where the drug goes after topical delivery.

Comments:

1) The authors note that IOP is reduced by 2.4 +/- 0.4 mm Hg and 14.1 +/- 1% compared to the other eye of the same rabbit. While this is nice, what was the IOP compared to the baseline IOP in the eye that was actually treated? In other words, what was the magnitude and percent of IOP reduction in the eye that was treated, pre- and post-? Getting data from both eyes (pre- and post- drug or vehicle) controls for more confounders.

2)While the Cmax for CKLP1 and levcromakalim may not have been significantly different in the 1- to 8-day experiments (Table 2), it appears that the Tmax might be? Do that calculation and include/implement. It is interesting to me because it says that the loading is faster. Makes sense given that the drop has been there for 7 days prior. The AUCs too.

3)If chromakalim (which is broken down to a dex- and lev-type) can lower IOP (citation 13), what is the advantage or need for CKLP1? Because it is already the lev-type? If one understood the breakdown of chromakalim and ratio of dex- and lev- that is produced, all one has to do is to upscale the dose of chromakalim to get more lev-. Further, the dex- had some IOP lowering effect too (citation 13). Unless, the dex- has some potential side-effect?

4)For the topical experiments, it is slightly strange that absolutely no CKLP1 is found in the aqueous and vit.

The authors suggest that testing the levels 24 hours after the last drop may have caused the aqueous turnover to make the drug leave the eyes (lines 285-298). I have a few thoughts.

-First, topical drugs have a much more direct route to the systemic body than aqueous outflow. Topical drugs simply enter the nasolacrimal duct system (like how we taste our salty tears when we cry). The drug can then be picked up by the blood vessels in the oropharynx and nasal mucosal epithelium. This alone can explain presence of drugs in the blood stream after topical delivery in the short-term experiments. This is also an issue for comment #5 below.

-Second, maybe the drug does not even have to get into the eye. The authors maybe testing the aqueous and vitreous under the assumption that that drugs must move into the eye to impact IOP-governing mechanisms. However, as the authors note, these drugs are supposed to effect the distal outflow pathways (lines 59-60). Thus, could the drug just penetrate into the scleral tissue and directly impact outflow as opposed to entering the anterior chamber first to do so?

-Third, the methods involve taking the aqueous/vit/plasma, spinning it down and testing the free fractions. What if CKLP1 is pigment bound? Beta blockers can be. In this case, the iris may have absorbed it all. Can we test for drug in iris? Try homogenization iris followed by a fast spin and testing what is in the sup.

-Fourth, what if there is differential protein binding such that the drug in the vitreous cavity was actually stuck with the vitreous that had not undergone syneresis? To address this, the pellet could be collagenase treated and spun again with the sup tested.

-Lastly, maybe the phosphatases are so fast that all the CKLP1 was metabolized.

5)Then for the topical experiments, it is strange that after 90 days, no levels were detected in the plasma. This is quite strange because both drugs were detected systemically after the shorter experiments at the 24-hour time-point. Further in 2 of 6 animals, no levcromakalim was seen in the aqueous while in 3 of 6 animals no levcromakalim was seen in the vitreous. This was unlike the shorter topical experiments were levcromakalim was more consistently detectable. Thus, the drug has to be somewhere. The renal/liver data suggests some elimination mechanism but this seems not enough. Since the drops applied on day 88 and 89 aren’t enough to show drug in the plasma, either the clearance method is revved up or there a depot where the drug is just accumulating. My feeling is that the drug is going somewhere that we are not recognizing. As above, I would look for the drug in other ocular tissues like the actual vitreous itself (you are only testing the liquified portion) and the iris. Also, in the blood fraction, test for binding. One could repeat the 90-day experiment, collect the blood and test for the drug in the packed RBC portion (after the spin). Alternatively, just test for in vitro binding by getting rabbit whole blood, mixing it with drug, waiting for a period of time, spinning the blood down and testing for the drug in the plasma vs. packed RPC portion. At least, this in vitro experiment could show if the drug can get into or onto RBCs and then help solve some of the mystery.

Minor issues:

1)lines 139… for this section, you must mean “for rabbits treated with CKLP1 for up to 8 days..” as opposed to just 8 days since some rabbits received treatment for 1- and 4-days.

2)lines 301: levcromakalim cannot be “consistently” identified if it is only seen in 3 of 6 rabbits for the vitreous and in 4 of 6 rabbits in the aqueous.

**********

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 to be viewed.]

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 us at figures@plos.org. Please note that Supporting Information files do not need this step.

Pharmacological and pharmacokinetic profile of the novel ocular hypotensive prodrug CKLP1 in Dutch-belted pigmented rabbits

PONE-D-20-00899R1

Dear Dr. Fautsch,

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.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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.

With kind regards,

Ted S Acott, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

concerns addressed

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

**********

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: (No Response)

Reviewer #2: Yes

**********

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

Reviewer #1: (No Response)

Reviewer #2: 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 Response)

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: (No Response)

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: (No Response)

Reviewer #2: The authors have attempted additional statistical analyses that I requested.

The authors have explained my misunderstanding of the drug

The authors have expanded their discussion as to alternative ways the drug could enter the body and given a better explanation as to why levcromakalim was only seen in 3 of 6 animals

**********

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