Implications of nasal delivery of bromelain on its pharmacokinetics, tissue distribution and pharmacodynamic profile—A preclinical study

Manu Sharma; Namita Gupta; Easha Pandey

doi:10.1371/journal.pone.0277849

Peer Review History

Original SubmissionJune 5, 2022
25 Jul 2022 Decision Letter - Lisa Ann Tell, Editor PONE-D-22-16222Implications of nasal delivery of bromelain on its pharmacokinetics, tissue distribution and pharmacodynamic profilePLOS ONE Dear Dr. Sharma, 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 Sep 08 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. 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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. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Additional Editor Comments: Dear Authors: Please make modifications to this manuscript to indicate that it is a preliminary study. The number of animal subjects overall and animal subjects per time point are small which doesn't really allow for assessment of differences. Please address this with your revision. [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: Yes 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: The PK and tissue distribution of bromelain were conducted and compared in rats following oral and nasal administration, and the pharmacodynamic profile was determined in guinea pigs. The present study is within the scope of PLOS ONE. HOWEVER, the writing elements need further improvement to enhance the quality of the manuscript, and copy editing is beyond the scope of a reviewer’s charge. The authors should pay more attention to further polishing this manuscript. Some of my specific comments are listed below: 1. The animals used in this study (rats and guinea pigs) should be added to both the title and abstract. Otherwise, readers will be misled into thinking that this study was conducted on human patients. 2. 2.3. Pharmacokinetic studies: To this reviewer’s knowledge, six rats were not enough in a PK study. Additionally, where were the blood samples collected? How many milliliters of blood were collected each time? All these details should be given in the revision. 3. Three animals per group were sacrificed at 0.5, 1, 2, 4 and 6 h post dosing. Likewise, three rats per time point were not sufficient. These data may not be sufficient to describe inter-individual differences. 4. Animals were exposed to an aerosol of … The authors should pay more attention to English. 5. Table 1: Which parameters were statistically significantly different (P<0.05)? 6. Figure 1B: This reviewer suggests the authors change the style of Figure 1B to Figure 1A. Reviewer #2: Abstract – The meaning of “unforeseeable prophylactic hypersensitivity due to its denaturation or agglomeration hinders its oral use” is unclear. Section 2.4 – For the oral dose of bromelain solution (40 mg/kg), what is the volume of administration? Is the drug in a solution or in a suspension? What is the vehicle of the solution? Is there any pH adjustment? The same questions can be applied to the nasal dose solution (10 mg/kg). How is the nasal dose administered? Are the animals constrained in a chamber? Is the nasal dose administered over a duration? Table 1, the increased in AUC looks like to be caused by a decrease in elimination? It may not be due to increase in bioavailability alone. The improved in efficacy may be due to higher drug concentration in the lung at 0.5 hour. Figure 1 – it looks like the substantial difference in the lung concentration is only observed at 0.5 hour. In Figure 1, labelling with “A” and “B” and “a” and “b” is confusing. ****** 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: Fan Yang 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. https://doi.org/10.1371/journal.pone.0277849.r001
Revision 1
8 Sep 2022 Author Response Academic Editor Comments Comment 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 Response: We ensure that our revised manuscript meets PLOS ONE’s style requirements, including those for file naming. Comment 2: To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering. Response: Additional information regarding the experiments involving animals have been included in Method section in detail in revised manuscript as per suggestion made by Academic Editor. Comment 3: Please make modifications to this manuscript to indicate that it is a preliminary study. The number of animal subjects overall and animal subjects per time point are small which doesn't really allow for assessment of differences. Please address this with your revision. Response: As per editor’s suggestion we have made changes in manuscript title and content to indicate that it is a preliminary study. We have performed further studies to derive data on sufficiently large number of animal subjects overall and animal subjects per time point for assessment of differences. The results have been included in revised manuscript. Response to reviewers’ comments Reviewer #1: The PK and tissue distribution of bromelain were conducted and compared in rats following oral and nasal administration, and the pharmacodynamic profile was determined in guinea pigs. The present study is within the scope of PLOS ONE. HOWEVER, the writing elements need further improvement to enhance the quality of the manuscript, and copy editing is beyond the scope of a reviewer’s charge. The authors should pay more attention to further polishing this manuscript. Some of my specific comments are listed below: Comment 1: The animals used in this study (rats and guinea pigs) should be added to both the title and abstract. Otherwise, readers will be misled into thinking that this study was conducted on human patients. Response: In order to prevent the wrong conception about the study by readers, correction in the title and abstract has been made as per reviewer’s suggestion. Title has been changed to “Implications of nasal delivery of bromelain on its pharmacokinetics, tissue distribution and pharmacodynamic profile- a preclinical study.” Similarly, changes has been made in abstract in revised manuscript i.e., “The present study investigated the preclinical prospects of nasal administration of bromelain on systemic bioavailability, tissue distribution and it’s in vivo anti-histaminic, bronchodilator and anti-asthmatic activity in animal models.” Comment 2: 2. 2.3. Pharmacokinetic studies: To this reviewer’s knowledge, six rats were not enough in a PK study. Additionally, where were the blood samples collected? How many milliliters of blood were collected each time? All these details should be given in the revision. Response: As per reviewer’s suggestion, twelve Wistar rats per group were taken in PK study. Blood samples (300 µl) were collected from rat tail vein in heparinised coated tubes at pre-dose (0.0), 0.5, 1, 2, 4 and 6 h post dose of bromelain solution. These details have been in incorporated in revised manuscript as “Twenty four Wistar rats were randomly distributed into two groups with twelve animals in each group. Animals of group I received bromelain solution orally (400 µl, 40 mg/Kg body weight) by oral gavage while second group received 30 µl, bromelain solution (10 mg/ Kg body weight) in each nostril in 30 s with the help of micropipette with 0.1 mm internal diameter at delivery place. Blood samples (300 µl) were collected from rat tail vein in heparinised coated tubes at pre-dose (0.0), 0.5, 1, 2, 4 and 6 h post dose of bromelain solution. Comment 3: Three animals per group were sacrificed at 0.5, 1, 2, 4 and 6 h post dosing. Likewise, three rats per time point were not sufficient. These data may not be sufficient to describe inter-individual differences. Response: As per reviewer’s suggestion, number of animals was increased to six at each time point i.e., six animals per group were sacrificed at 0.5, 1, 2, 4 and 6 h post dosing. The results of tissue distribution study at each time point is average of six readings obtained from six animals sacrificed at respective time. The corrections have been incorporated in revised manuscript under Tissue distribution study as “Sixty healthy wistar rats were randomized into two groups (n= 30). Rats of group I received bromelain solution orally (40 mg/kg body weight) while group II received bromelain solution nasally (10 mg/kg body weight) in each nostril with the help of micropipette with 0.1 mm internal diameter respectively. Six animals per group were sacrificed at 0.5, 1, 2, 4 and 6 h post dosing. Comment 4: Animals were exposed to an aerosol of … The authors should pay more attention to English. Response: As per reviewer’s suggestion, correction has been incorporated in revised manuscript i.e., “Animals were challenged with histamine dihydrochloride aerosol (0.5% w/v) using a nebulizer at a pressure of 300 mm Hg in an air tight Plexiglass chamber (24 × 14 × 24 cm) for 1 min. Consequently after histamine exposure, animals were closely observed to record the pre-convulsive dyspnea time and convulsion time for 9 minutes.” Comment 5: Table 1: Which parameters were statistically significantly different (P<0.05)? Response: All the pharmacokinetic parameters like Cmax, Tmax, ke, t1/2, MRT and AUC0-24h were statistically significantly different (p<0.05). The same has been highlighted in Table 1. Comment 6: Figure 1B: This reviewer suggests the authors change the style of Figure 1B to Figure 1A. Response: As per reviewer’s suggestion, style of Figure 1B has been changed to Figure 1A. Reviewer #2: Comment 1: Abstract – The meaning of “unforeseeable prophylactic hypersensitivity due to its denaturation or agglomeration hinders its oral use” is unclear. Response: In order to have more clarity changes has been made in abstract as per reviewer’s suggestion. The changes made are presented in revised manuscript as “However, poor stability in gastric milieu, high dose and immunogenicity associated with prolonged use hinders its oral use.” Comment 2: Section 2.4 – For the oral dose of bromelain solution (40 mg/kg), what is the volume of administration? Is the drug in a solution or in a suspension? What is the vehicle of the solution? Is there any pH adjustment? The same questions can be applied to the nasal dose solution (10 mg/kg). How is the nasal dose administered? Are the animals constrained in a chamber? Is the nasal dose administered over a duration? Response: For the oral dose of bromelain solution (40 mg/kg), 400 µl of bromelain solution was administered by oral gavage. The vehicle of solution was distilled water. No pH adjustment of bromelain solution was done during study. 30 µl of bromelain solution at a dose of 10 mg/Kg was administered in each nostril in 30 s with the help of micropipette with 0.1 mm internal diameter at delivery place. Nasal dose was administered using intransal grip, each rat was restrained twice and held with their neck parallel to the floor while administering nasal dose. As per reviewer’s suggestion changes have been made in manuscript and represented as “Animals of group I received bromelain solution orally (400 µl, 40 mg/Kg body weight) by oral gavage while second group received nasally (30 µl, 10 mg/ Kg body weight) in each nostril in 30 s with the help of micropipette with 0.1 mm internal diameter at delivery place. Blood samples (300 µl) were collected from rat tail vein in heparinised coated tubes at pre-dose (0.0), 0.5, 1, 2, 4 and 6 h post dose of bromelain solution. Nasal dose was administered using intransal grip, each rat was restrained twice and held with their neck parallel to the floor while administering nasal dose. Comment 3: Table 1, the increased in AUC looks like to be caused by a decrease in elimination? It may not be due to increase in bioavailability alone. The improved in efficacy may be due to higher drug concentration in the lung at 0.5 hour. Response: The decrease in elimination rate constant increased the t1/2 and mean residence time which contributed to increase in AUC i.e., systemic bioavailability. Nasal administration improved the drug concentration in lungs which might have improved therapeutic efficacy of bromelain. Comment 4: Figure 1 – it looks like the substantial difference in the lung concentration is only observed at 0.5 hour. In Figure 1, labelling with “A” and “B” and “a” and “b” is confusing. Response: As per reviewer’s suggestion, labeling has been changed in Figure 1. Attachments Attachment Submitted filename: Response to reviewers.doc https://doi.org/10.1371/journal.pone.0277849.r002
4 Nov 2022 Decision Letter - Lisa Ann Tell, Editor Implications of nasal delivery of bromelain on its pharmacokinetics, tissue distribution and pharmacodynamic profile- a preclinical study PONE-D-22-16222R1 Dear Dr. Sharma, 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, Lisa Ann Tell 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: 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: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: 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 ****** 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 author has fully addressed my previous comments. I think this manuscript is acceptable in its current state. ****** 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: Yes: Fan Yang ******** https://doi.org/10.1371/journal.pone.0277849.r003
Formally Accepted
10 Nov 2022 Acceptance Letter - Lisa Ann Tell, Editor PONE-D-22-16222R1 Implications of nasal delivery of bromelain on its pharmacokinetics, tissue distribution and pharmacodynamic profile- a preclinical study Dear Dr. Sharma: 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. Lisa Ann Tell Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0277849.r004

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