Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis Using a Mixure of Bromelain, Papain and Ficin Supplement

Round 1

Reviewer 1 Report

The Authors have written an interesting article; some paragraphs might be improved.

-The Authors should provide a detailed References list for what has been written in the Manuscript, without any reference to the literature (some examples: A study demonstrated that patients with isolated PVD, VH and ocular floaters at initial presentation needed to be rescheduled for a follow-up visit; Only 11% of subjects’ eyes should show spontaneous dissociation over a mean of 5 years; some 64% of victim’s eyes lost at least 2 Snellen lines after several years; RD would occur in 2.4% and 92% of eyes developed into cataract within 3 years after vitrectomy; In our past study; According to previous studies, acute onset of floaters and even PVD would develop into retinal break within 6 weeks..)

-How was the "special enteric release capsule" developed ? Please explain in further detail.

-Please explain in further detail how and when OCT was used in the study.

-The Introduction section is very long and many paragraphs might be moved in the Results and Discussion section.

-Was Best Corrected Visual Acuity evaluated ? If not, this should be reported as a major limitation of the study.

-In the Abstract it is said that the study was performed between April 2017 and December 2017, in the Materials and Methods between April and December 2016. Please specify what is the correct period.

-Reference 48 is reported in the manuscript but there are only 43 References. Moreover, in the manuscript the phrase "Previously, we reported that vitreous floaters could be significantly absorbed after a 3-month pineapple supplement" does not reference a study by the Authors (Reference 43)

-The final outcome in experiment 2 placebo group is 98% in page 7 and 95% in page 8. Please specify which is the correct percentage. LPG group in page 5 is said to be "4 cases", I think 40 is the correct number.

- "No serious side effects" are reported during treatment, are there minor side effects to be reported ?

- A comprehensive english revision should be performed (bromelian; disappeared rates; tretment; resluts; victims; floater0; peroid; All 190 patients all took; turn the head try; phadodonesis, etilogy, bsed..). What is "eclectic microscopy" ? And collagen VX ?

- Please find more appropriate terms than "useless vitreous fibrils", "that it is the first report and publication in the world." Moreover, as vitreous floaters from blood clots and other opacities are still removed by laser vitreolysis and PPV, do not use the term "in the past" before the phrase "Now, however, taking the capsules of mixed –fruit-enzyme..."

Author Response

Dear reviewer,

 

Thank you for the precious suggestions on the manuscript, “Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin Supplement,” and we have made substantial revisions to the manuscript based on the reviewer’ comments, as the details below.

• The Authors have written an interesting article; some paragraphs might be improved.

-The Authors should provide a detailed References list for what has been written in the Manuscript, without any reference to the literature (some examples: A study demonstrated that patients with isolated PVD, VH and ocular floaters at initial presentation needed to be rescheduled for a follow-up visit; Only 11% of subjects’ eyes should show spontaneous dissociation over a mean of 5 years; some 64% of victim’s eyes lost at least 2 Snellen lines after several years; RD would occur in 2.4% and 92% of eyes developed into cataract within 3 years after vitrectomy; In our past study; According to previous studies, acute onset of floaters and even PVD would develop into retinal break within 6 weeks..)

Response: We have rewritten the manuscript and provided the proper references.

• How was the "special enteric release capsule" developed ? Please explain in further detail.

Response:The components of the slow released capsule were HPMC (hydroxyl propyl methylcellulose) and pectin. HPMC is a semisynthetic, inert, viscoelastic polymer used as eye drops, as well as an excipient and controlled-delivery component in oral medicaments, which can be found in a variety of commercial products.Pectin is as a gelling agent, thickening agent and stabilizer. In capsule, pectin may resist the gastric acid and prevent the fruit enzymes in the stomach.

• Please explain in further detail how and when OCT was used in the study.

Response: First, we select the patient with ocular floater by using the indirect ophthalmoscopy (Photogram picture of fundus). If failed, the B-scan was used to screen this subject. The OCT instrument was the final step to check the abnormal image.

The indirect ophthalmoscopy, B-scan and OCT were used to screen the ocular floaters cases.

• The Introduction section is very long and many paragraphs might be moved in the Results and Discussion section.

Response:We have rewritten the manuscript.

• Was Best Corrected Visual Acuity evaluated ? If not, this should be reported as a major limitation of the study.

Response: In the end of each month, we inspected the variation of patients' floaters and recorded the visual acuity. The change of the best corrected visual acuity (BCVA) of the patient may be caused by the  function of mixture of fruit enzymes.

• In the Abstract it is said that the study was performed between April 2017 and December 2017, in the Materials and Methods between April and December 2016. Please specify what is the correct period.

Response: Our research was scheduled in Southern Taiwan between April 2017 and December 2017.  

• Reference 48 is reported in the manuscript but there are only 43 References. Moreover, in the manuscript the phrase "Previously, we reported that vitreous floaters could be significantly absorbed after a 3-month pineapple supplement" does not reference a study by the Authors (Reference 43)

Response:

We have corrected this mistake.

• The final outcome in experiment 2 placebo group is 98% in page 7 and 95% in page 8. Please specify which is the correct percentage. LPG group in page 5 is said to be "4 cases", I think 40 is the correct number.

Response: The final outcome in experiment 2 placebo group is 95%,and the exact number was 40.

• "No serious side effects" are reported during treatment, are there minor side effects to be reported ?

Response: No serious side effects were found such as the hives, skin rash, anaphylactic shock from allergic reaction.Even, we didn't find  minor side effects in this experiment.

• A comprehensive english revision should be performed (bromelian; disappeared rates; tretment; resluts; victims; floater0; peroid; All 190 patients all took; turn the head try; phadodonesis, etilogy, bsed..). What is "eclectic microscopy" ? And collagen VX ?Please find more appropriate terms than "useless vitreous fibrils", "that it is the first report and publication in the world." Moreover, as vitreous floaters from blood clots and other opacities are still removed by laser vitreolysis and PPV, do not use the term "in the past" before the phrase "Now, however, taking the capsules of mixed –fruit-enzyme..."

Response: The  manuscript  have be revised by native English speaker. Useless vitreous fibrils have be replaced by futile vitreous fibrils.

Kind regards

With above responses and substantial revisions, please kindly consider its publication in Applied sciences. Thank you for your kind consideration and assistance.

 

Sincerely,

 

Chi-Ting Horng M.D., Ph.D.

Mail: [email protected]

Reviewer 2 Report

The proposed idea is interesting and innovative. However, there is still work to improve to be published. I recommend:

• Summarize the introduction in a better way, being more concise, since it is overloaded with information (part of the information that would be better in the discussion) that makes it lose interest and focus of the work. In that sense, authors would also structure the introductory text in a more fluid way.
• Pay attention that double spaces appear between the words in the text. Also the formatting of the paragraphs, since lines appear with greater separation than others. Furthermore, the conclusions have a different line spacing than the results, authors must maintain a criteria in the format.
• In the methodology there are unnecessary explanations, it is a section that must be objective and say what was done, not give the "why". In addition, it would separate into subsections for a better reading.
• The figures must appear after being cited in the text, not before, as the authors did.
• In the text authors talk about correlation between at the higher dose of enzyme intake and the higher disappeared rates, but without specifying anywhere the type of statistical correlation analysis performed.
• Authors have to review the use of significant figures, which in some cases is not well applied, for example "47.9 ± 2.5 years old".
• Review the style of bibliographic citations, being consistent with those commonly used in the Journal.

Kind regards

Author Response

Dear reviewer,

Thank you for the precious suggestions on the manuscript, “Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin Supplement,” and we have made substantial revisions to the manuscript based on the reviewer’ comments, as the details below.

The proposed idea is interesting and innovative. However, there is still work to improve to be published. I recommend:

• Summarize the introduction in a better way, being more concise, since it is overloaded with information (part of the information that would be better in the discussion) that makes it lose interest and focus of the work. In that sense, authors would also structure the introductory text in a more fluid way.

Response:

Thank you for your kindly reminder. We have improved these mistakes in the introduction section. We have summarized the introduction in the manuscript.

• Pay attention that double spaces appear between the words in the text. Also the formatting of the paragraphs, since lines appear with greater separation than others. Furthermore, the conclusions have a different line spacing than the results, authors must maintain a criteria in the format.

Response:

Thank you for your suggestion. From the analysis results before and after treatment, the pair T-test assay found that there is a significant improvement effect after taking fruit enzymes for 3 months. The difference was compared by paired t-test in experiment 1. We further compared the results between placebo, LPG, MPG, and HPG at baseline and during the whole 3-months by Scheffee test. Besides, the results between the 4 groups were analyzed by Williams’ test in experiment 2. Moreover, P value < 0.05 was considered statistically significant.In the methodology there are unnecessary explanations, it is a section that must be objective and say what was done, not give the "why". In addition, it would separate into subsections for a better reading.

Response:

Thank you for your kindly reminder. We have improved these mistakes in the materials and methods section.

• The figures must appear after being cited in the text, not before, as the authors did.

Response:

Thank you for your kindly reminder. We have added the figures cited in the whole manuscript.

• In the text authors talk about correlation between at the higher dose of enzyme intake and the higher disappeared rates, but without specifying anywhere the type of statistical correlation analysis performed.

Response:

The difference was compared by paired t-test in experiment 1. We further compared the results between placebo, LPG, MPG, and HPG at baseline and during the whole 3-months by Scheffee test. Besides, the results between the 4 groups were analyzed by Williams’ test in experiment 2. Moreover, P value < 0.05 was considered statistically significant.

• Authors have to review the use of significant figures, which in some cases is not well applied, for example "47.9 ± 2.5 years old".

Response:

• Thank you for your kindly reminder. We have exchange the "47.9 ± 2.5 years old" to "48 ± 3 years old".

Kind regards

With above responses and substantial revisions, please kindly consider its publication in Applied sciences. Thank you for your kind consideration and assistance.

 

Sincerely,

 

Chi-Ting Horng M.D., Ph.D.

Mail: [email protected]

Reviewer 3 Report

This paper cannot be review as it is not adhering the guidelines of the journal

First, no subsection and clear methodology and results. Please, major revisions are needed here.

The introduction is 3 pages, this has to be shortened by a minimum of half.

The methodology has to be checked very carefully and details all the sections including the statistics that is the important part.

The presentation of the results should be given in an accurate manner before you discuss them according to the literature.

The conclusion needs major revision. Please check very carefully and avoid citing references.

The references are not well cited in the manuscript, please check and use [] instead of ().

Author Response

Dear reviewer,

  Thank you for the precious suggestions on the manuscript, “Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin Supplement,” and we have made substantial revisions to the manuscript based on the reviewer’ comments, as the details below.

1. This paper cannot be review as it is not adhering the guidelines of the journal.

Response:

Thank you for your kindly reminder. We have improved the manuscript according by reviewer suggestion and guidelines of the journal.

2. First, no subsection and clear methodology and results. Please, major revisions are needed here.

Response:

Thank you for your kindly suggestion. We have added the subsection in methodology and results.

3. The introduction is 3 pages, this has to be shortened by a minimum of half.

Response:

Thank you for your kindly suggestion. We have shortened and summarized the section of introduction.

4. The methodology has to be checked very carefully and details all the sections including the statistics that is the important part.

Response:

Thank you for your kindly suggestion. We have checked the section of methodology and statistics carefully.

From the analysis results before and after treatment, the pair T-test assay found that there is a significant improvement effect after taking fruit enzymes for 3 months. The difference was compared by paired t-test in experiment 1. We further compared the results between placebo, LPG, MPG, and HPG at baseline and during the whole 3-months by Scheffee test. Besides, the results between the 4 groups were analyzed by Williams’ test in experiment 2. Moreover, P value < 0.05 was considered statistically significant.

5. The presentation of the results should be given in an accurate manner before you discuss them according to the literature.

Response:

Thank you for your kindly suggestion. We have improved the section of results and discussion.

6. The conclusion needs major revision. Please check very carefully and avoid citing references.

Response:

Thank you for your kindly suggestion. We have revised the section of conclusion.

 

7. The references are not well cited in the manuscript, please check and use [] instead of ().

Response: The references citation is improved in the manuscript.

 

Kind regards

With above responses and substantial revisions, please kindly consider its publication in Applied sciences. Thank you for your kind consideration and assistance.

 

Sincerely,

 

Chi-Ting Horng M.D., Ph.D.

Mail: [email protected]

Round 2

Reviewer 1 Report

The Authors have revised their manuscript and the effort has been appreciated; some minor revisions are still requested.

-A revision of the References should be performed again; some paragraphs still do not have any reference to the literature (some examples: lines 78-79; 309-310..) and some others do not have a correct reference (some examples: lines 421-422 and reference 39, which is not a study by the Authors; lines 45-47...) 

-The fact that Best Corrected Visual Acuity was evaluated at the end of every month must be clearly written into the manuscript (even if combined data of all these patients is not the purpose of the Authors' study)

-The fact that the components of the slow released capsule were HPMC (hydroxyl propyl methylcellulose) and pectin should be stated in the Materials section

-There are still some English corrections that may be performed: bromelian (lines 13 and  29), tretment (line 22), resluts (line 24)..Moreover, as said in the previous Comments and Suggestions for Authors, eclectic microscopy does not exist and line 434 can still be ok without the phrase "that it is the first report and publication in the world."

Author Response

Dear reviewer,

 

Thank you again for the precious suggestions on the manuscript, “Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin Supplement,” and we have made substantial revisions to the manuscript based on the reviewer’ comments, as the details below.

The Authors have revised their manuscript and the effort has been appreciated; some minor revisions are still requested.

-A revision of the References should be performed again; some paragraphs still do not have any reference to the literature (some examples: lines 78-79; 309-310..) and some others do not have a correct reference (some examples: lines 421-422 and reference 39, which is not a study by the Authors; lines 45-47...) 

Response: The references of the manuscript have be carefully revised again. We haved cited reference [ 14 ] in lines 78-79; [ 15] in line 80, and reference [ 25] in lines 311-312. The correct reference [41] have be cited in lines 428-429, and reference [ 2] in lines 47-48.

-The fact that Best Corrected Visual Acuity was evaluated at the end of every month must be clearly written into the manuscript (even if combined data of all these patients is not the purpose of the Authors' study)

Response:

We have added the sentence ” We also evaluated the best corrected visual acuity (BCVA) of the subjects at the end of every month. “ in lines 143-144 of Experimental design and Laboratory examination section.

We have added the sentence “The best corrected visual acuity (BCVA) was improved, but it was not statistically significant difference.” in lines 161-162 of Patient characteristics section.

-The fact that the components of the slow released capsule were HPMC (hydroxyl propyl methylcellulose) and pectin should be stated in the Materials section

Response: We have added the sentence “The components of the slow released capsule were hydroxyl propyl methylcellulose (HPMC) and pectin.” in lines 105-106 of Materials section.

-There are still some English corrections that may be performed: bromelian (lines 13 and  29), tretment (line 22), resluts (line 24)..Moreover, as said in the previous Comments and Suggestions for Authors, eclectic microscopy does not exist and line 434 can still be ok without the phrase "that it is the first report and publication in the world."

Response: We have correted “bromelian” in lines 13 and 29, “treatment” in line 22, and “results” in line 24. The eclectic microscopy have be correted as slit lamp examination. We have deleted the sentence “that it is the first report and publication in the world.” in lines 441-442.

Kind regards

 

With above responses and substantial revisions, please kindly consider its publication in Applied sciences. Thank you for your kind consideration and assistance.

 

Sincerely,

 

Chi-Ting Horng M.D., Ph.D.

Mail: [email protected]

Reviewer 2 Report

I congratulate the authors for the work to improve the manuscript, considering the suggestions made by the reviewers. I acknowledge the article to be published in its current state.

 

Best regards.

Author Response

Dear reviewer,

Thank you again for the precious suggestions on the manuscript and accepted for publication.

Sincerely,

Chi-Ting Horng M.D., Ph.D.

Mail: [email protected]

 

Reviewer 3 Report

This paper has serious luck in the experimental desing and conditions of use of the enzymes. No novelty. Ethical approval is needed.

Author Response

Dear reviewer,

 

Thank you again for the precious suggestions on the manuscript, “Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin Supplement,” and we have made substantial revisions to the manuscript based on the reviewer’ comments, as the details below.

 

This paper has serious luck in the experimental desing and conditions of use of the enzymes. No novelty. Ethical approval is needed.

Response: Thank you for for the precious suggestions. We have provided the Ethical approval to MDPI.

 

Kind regards

 

With above responses and substantial revisions, please kindly consider its publication in Applied sciences. Thank you for your kind consideration and assistance.

 

Sincerely,

 

Chi-Ting Horng M.D., Ph.D.

Mail: [email protected]

Round 3

Reviewer 3 Report

New comments on the paper applsci-903381

1. Change the title as follow: "Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis Using a mixture of plant cysteine proteases"
2. line 48 - 49: remove the sentence Our research was scheduled in Southern Taiwan between April 2017 and 48 December 2017
3. Line 219, a reference is needed
4. line 334 and in all the manuscript, change fruit enzymes by plant proteases.
5. Authors are asked to present in a Table the characteristics and origin of the plant cysteine proteases used. Some biochemical parameters are needed. Please support this with some data of their targeted proteins. Some studies can be cited or used to build this new Table (10.1016/j.ijbiomac.2019.10.048; 10.1016/j.seppur.2014.05.050; 10.1016/j.ijbiomac.2020.06.144).
6. For M&M section, please add numbering for the sub-sections.
7. For all the Figures, the captions and footnotes are weak. More information are needed. Please try to explain as much as possible the graphs. Also, try to highlight on the graphs the differences.
8. Table 2: the use of protein content is false. Please and in the whole manuscript try to give the activity units of the enzymes. Amount of the protein is not worthy in this type of studies. This is a MAJOR comment.
9. The discussion is very long and not structured. Please remove a minimum of one page. Add sub-titles to better follow the discussion. More references from the literature are needed.
10. The conclusion needs further revision. What are the future guidelines? How this study can help move forward in this topic?

Author Response

Dear reviewer,

Thank you again for the precious suggestions on the manuscript, “Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin Supplement,” and we have made substantial revisions to the manuscript based on the reviewer’ comments, as the details below.

1. Change the title as follow: "Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis Using a mixture of plant cysteine proteases"

Response: Thank you for the comment. We have changed the title as follow. “Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis Using a Mixture of Bromelain, Papain and Ficin Supplement” The three fruit enzymes were used in this study, it should be specifically marked in the title.

2.line 48 - 49: remove the sentence Our research was scheduled in Southern Taiwan between April 2017 and 48 December 2017

Response: Thank you for the comment. lines 48-49 did not show the sentence “Our research was scheduled in Southern Taiwan between April 2017 and 48 December 2017”

3. Line 219, a reference is needed

Response: Thank you for the comment. The references of the manuscript have be carefully revised again.

4. line 334 and in all the manuscript, change fruit enzymes by plant proteases.

Response: Thank you for the comment. After several dsiscussions with coauthors, we think that the fruit enzymes are more suitable than plant proteases in this study.

5. Authors are asked to present in a Table the characteristics and origin of the plant cysteine proteases used. Some biochemical parameters are needed. Please support this with some data of their targeted proteins. Some studies can be cited or used to build this new Table (10.1016/j.ijbiomac.2019.10.048; 10.1016/j.seppur.2014.05.050; 10.1016/j.ijbiomac.2020.06.144).

Response:

Thank you for the comment. We have added “The molecular weight and isoelectric point (MW, PI) are (33000, 9.55), (23406, 8.75) and (26000, 9.0) for bromelain, papain and  ficin, respectively [18-20].” In lines 105-106. The suggested references were also be cited as ref. [18-20]

18. Kahina Hafid, James John, Taha Mansour Sayah , Rubén Domínguez , Samira Becila , Melisa Lamri , Amira Leila Dib , José M Lorenzo , Mohammed Gagaoua : One-step recovery of latex papain from Carica papaya using three phase partitioning and its use as milk-clotting and meat-tenderizing agent. Int J Biol Macromol. 2020;146:798-810. doi:10.1016/j.ijbiomac.2019.10.048

19. MohammedGagaoua,NawelBoucherba, Amel Bouanane-Darenfed, Ferhat Ziane, Sabrina Nait-Rabah, Kahina Hafid, Hiba-Ryma Boudechicha: Three-phase partitioning as an efficient method for the purification and recovery of ficin from Mediterranean fig (Ficus carica L.) latex. Separation and Purification Technology. 2014, 132: 461-467doi:10.1016/j.seppur.2014.05.050
20. Morellon-Sterling R, El-Siar H, Tavano OL, Berenguer-Murcia Á, Fernández-Lafuente R. Ficin: A protease extract with relevance in biotechnology and biocatalysis [published online ahead of print, 2020 Jun 20]. Int J Biol Macromol. 2020;162:394-404. doi:10.1016/j.ijbiomac.2020.06.144

 

6. For M&M section, please add numbering for the sub-sections.

Response:

Thank you for the comment. We have added numbering for the sub-secions of M&M and R&D sections.

7. For all the Figures, the captions and footnotes are weak. More information are needed. Please try to explain as much as possible the graphs. Also, try to highlight on the graphs the differences.

Response:

Thank you for the comment. We have revised all the figure captions, and footnotes. We have also marked the differences of the figures.

 

8.Table 2: the use of protein content is false. Please and in the whole manuscript try to give the activity units of the enzymes. Amount of the protein is not worthy in this type of studies. This is a MAJOR comment.

Response: Thank you for the comment. The three fruit enzymes were obtained commercially and described in section 2.1. In this study,we did provide the fruit enzymes capsules to subjects, and evalute the effect of fruit enzymes on the change of various floaters by clinical technique such as B-Scan and OCT.  The amount of enzyme was more easier to be understood for clinical research. We didn’t calculate the enzymes activity in this study, we will try to calculate the enzymes activity in the future work.

 

9.The discussion is very long and not structured. Please remove a minimum of one page. Add sub-titles to better follow the discussion. More references from the literature are needed.

Response: Thank you for the comment. We have modified the results and discussions again, and all the references were revised.

10. The conclusion needs further revision. What are the future guidelines? How this study can help move forward in this topic?

 Response: Thank you for the comment. The conclusion have been revised again.

Kind regards

With above responses and substantial revisions, please kindly consider its publication in Applied sciences. Thank you for your kind consideration and assistance.

 

Sincerely,

 

Chi-Ting Horng M.D., Ph.D.

Mail: [email protected]

 

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The Manuscript authored by Takeuchi entitled "Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin" is difficult to follow, quite confusing and not totally clear. It needs to be rewritten, portions of the study are not detailed and more evidences should be presented.

Authors investigated a mix of proteases (bromelian, papain and ficin) in capsules to treat symptomatic vitreous opacity (SVO) in patients that were clinically diagnosed with this condition in the eye. 280 patients were admitted in this clinical study.

According to authors’ findings, it seems that this treatment with fruit proteases may be efficient to treat SVO since some patients recovered and eyes were improved after. Controls were used, including placebo.

However, this manuscript does not fit with Polymers’ aims and scope. Fruit protaeases can be considered polymers, biological ones, naturally synthetized with amino acids as building blocks. But no further characterization of polymers are presented in the manuscript. These enzymes were acquired from companies and formulated in capsules. No studies of bioavailability were performed and presented.

Besides, how authors can guarantee that the enzymes would stay integer in the digestive system and gastrointestinal tract. Also, no evidences were showed demonstrating that the enzymes would be absorbed and reach vitreous liquid inside human eyes. And about ADME and the first pass effect?

Authors need to clarify why these experiments and controls were not used and considered. Discussion fails in giving more support to authors’ conclusion. I recommend extending some experiments by determining availability and absorption of these enzymes. These results will certainly improve a lot authors' discussion and conclusion.

The figures and table are well-distributed, presented in good graphical quality and include the main results authors found in this manuscript.

To finish this revision, besides these above mentioned suggestions, the manuscript needs a complete restructuring and it lacks in stronger evidences. This work might be an important contribution to our knowledge of fruit proteases as an efficient pharmaceutical for SVO. However, the manuscript needs more details as recommended herein.

In this format, it should not be considered for publication in Polymers, in fact, I recommend other journals with a more clinical scope.

Author Response

Dear reviewer,

 

Thank you for the precious suggestions on the manuscript, “Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin Supplement,” and we have made substantial revisions to the manuscript based on the reviewer’ comments, as the details below.

1. The Manuscript authored by Takeuchi entitled "Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin" is difficult to follow, quite confusing and not totally clear. It needs to be rewritten, portions of the study are not detailed and more evidences should be presented.

Response: The manuscript has been rewritten, and edited by a native English speaker.

2. Authors investigated a mix of proteases (bromelian, papain and ficin) in capsules to treat symptomatic vitreous opacity (SVO) in patients that were clinically diagnosed with this condition in the eye. 280 patients were admitted in this clinical study.According to authors’ findings, it seems that this treatment with fruit proteases may be efficient to treat SVO since some patients recovered and eyes were improved after. Controls were used, including placebo.However, this manuscript does not fit with Polymers’ aims and scope. Fruit protaeases can be considered polymers, biological ones, naturally synthetized with amino acids as building blocks. But no further characterization of polymers are presented in the manuscript. These enzymes were acquired from companies and formulated in capsules. No studies of bioavailability were performed and presented.

Response:

(1)The method of treating symptomatic vitreous opacity (SVO) from pharmacologic vitreolysis was only by intravitreal injection (IVI) of ocriplasmin (microplasmin) which was truncated form of human serine protease plasmin and only used in USA and EU. However, there are many adverse events from this procedure and treatment including intragenic cataract, glaucoma and even retinal detachment after repeated injection.

(2)Besides, the technique of IVI is uncomfortable for patients who sometimes hesitated to receive injection. Therefore, oral routine (e.g.: the mixed fruit enzymes in one capsule ) may have advantsges of cheap price, safty, convenience in the future.

(3)In our experiment, we also found that ficin could cut the links of hyaluronic acid which could be associated with the bioactivity of serine. The functions of ficin were similar to ocriplasmin which could be used to treat ocular opacity in clinics.      

3. Besides, how authors can guarantee that the enzymes would stay integer in the digestive system and gastrointestinal tract. Also, no evidences were showed demonstrating that the enzymes would be absorbed and reach vitreous liquid inside human eyes. And about ADME and the first pass effect?

Response:

(1) In this study, we developed the new therapy for pharmacologic vitreolysis. There are several different enzymes in three fruits including bromelain, papain and ficin which are made of various amino acids and other substances. The results indicated that the enzymes could cut and dissolve the pcollagen fibers and hyaluronic acid in the vitreous of human eyes.

(2) In experiment 1, 80 patients with one SVO decreased to 24 subjects (30.0%; 24 /80) (P < .05), and the rate of treatment success was 70% after 3 months. The 40 participants with multiple SVOs decreased to 11 patients (27.5%; 11/40) after the 3-month therapy (P < .05). The disappeared rate was 72.5% in the multiple-SVOs group (Table 1). In experiment 2, the final outcomes in the 4 groups with placebo, LPG, MPG, and HPG were 98%, 35%, 30%, and 25%, respectively (Figs. 1(a),1(b), 2(a), 2(b), 3(a) and 3(b)). In other words, the disappeared rates were 65%, 70%, and 75% of SVOs in LGP, MGP, and HGP, respectively, after 3- months of taking the mixed-fruit-enzyme supplements (P < .05) (Table 2). Hence, the results also show the dose-dependent correlation between higher dose of enzyme intake and higher disappeared rates; the higher doses of our capsules have a stronger hydrolysis ability for degrading SVOs. However, the ocular opacity in the placebo group did not vanish remarkably without the fruit enzymes (P > .05). Furthermore, a statistical test was performed to compare between the dose-dependent and placebo groups by Williams’ test. The results show that all of the percentages of disappeared rate in LPG, MPG and HPG are statistically significant when compared with placebo group (Table 2). Ocular examinations in this study found that the SVOs had been absorbed in the “successful” cases, thus, our capsules should enhance the functions of hydrolysis and lysis. Furthermore, we also found that SOVs from patients with diabetic retinopathy could be dissolved by fruit enzymes, and the mechanism of disappearance of VH may be primarily due to the anti-thrombotic function of papain. Therefore, we suggest that a combination of bromelain, papain, and ficin can easily dissolve and absorb ocular opacities.

 

 (3) We  have discussed with serval ophthalmologists and biochemistries on the limitation and difficulties in the human trials from ocular blood and ocular tissues. Although ADME , the first pass effect, and bio-availabilities from these fruit enzymes are very important in the fundamental science.However, in the clinical research, we carry on research by measuring some  parameters such as logMAR chart,  the non-dilated pupil ophthalmoscopy, ocular ultrasound and optical coherence tomography. 

(4) We used the method of Willams’test to evaluate the statistic difference between the placebo and the other dose-dependent groups. Thus, the more information we obtained is added in the pages 7-9 of the manuscript and described as below:

. Furthermore, a statistical test was performed to compare between the dose-dependent and placebo groups by Williams’ test. The results show that all of the percentages of disappeared rate in LPG, MPG and HPG are statistically significant when compared with placebo group (Table 2).  Ocular examinations in this study found that the SVOs had been absorbed in the “successful” cases, thus, our capsules should enhance the functions of hydrolysis and lysis. Furthermore, we also found that SOVs from patients with diabetic retinopathy could be dissolved by fruit enzymes, and the mechanism of disappearance of VH may be primarily due to the anti-thrombotic function of papain. Therefore, we suggest that a combination of bromelain, papain, and ficin can easily dissolve and absorb ocular opacities.

    An ocular floater or opacity is the result of opaque fragments floating in the vitreous. This study shows that most of the ocular floaters did not impact vision; and the numbers of floaters sometimes increased with age. Although the symptoms were minimal, they caused significant impairment in vision-related quality of life for some victims﹝24﹞. While the SOVs may occur suddenly and bother the subjects, patients might adapt to vitreous opacities over a 3-month period. SVOs can be associated with PVD with or without retinal breaks. Moreover, some ocular floaters may be caused by the solitary blood clots of various retinopathies, such as diabetic retinopathy and intraocular blood due to trauma.

                                                                                                    

 

 

 

 

 

Table 1: The changes in various floaters before and after taking the fruit protease supplements over a 3-month period

Time Number	Baseline	1st month	2nd month	3rd month
One   Multiple	80   40	78      37	68       30	24* (30.0%)   11* (27.5%)

   N= 190 eyes

1. Multiple floaters mean a patient with at least 2 ocular floaters in the visual field.
2. After treatment, multiple floaters may completely or incompletely disappear; therefore, multiple floaters would change to 1 or 0 floaters.
3. All 190 patients took one capsule twice daily, and every fruit protease capsule included 190 mg bromelain, 95 mg papain, and 95 mg ficin.

*4. The results at the baseline and after 3 months were compared by paired t-test, where a p value less than .05 means significant difference.

 

Table 2: The changes in ocular floaters before and after the fruit protease supplements 

over a 3-month period

Time Capsule daily	Baseline	1st month	2nd month	3rd month
none   1 capsule   2 capsules   3 capsules	40       40      40      40	40      38      35      34	40         33       32       30	38 (95.0%)   14* (35.0%)#   12* (30.0%)#   10* (25.0% )#

1. Group 1 (N= 40 eyes): no protease supplements were taken (placebo group). Group 2 (N= 40 eyes): all patients took 1 mixed protease capsule including 190 mg bromelain, 95 mg papain, and 95 mg ficin every day. Group 3 (N= 40 eyes): all patients took 2 capsules daily. Group 4 (N= 40 eyes): all patients took 3 capsules daily.

*2. The results at the baseline and after 3 months were compared by pair-t test, where a p value less than .05 means a significant difference.

^#3. We compared the results between the placebo and dose-dependent groups after 3-month therapies by Williams’ test, where a p value less than .05 means significant difference.

With above responses and substantial revisions, please kindly consider its publication in Polymers. Thank you for your kind consideration and assistance.

 

Sincerely,

 

Chi-Ting Horng M.D., Ph.D.

Mail: [email protected]

 

 

Author Response File: Author Response.docx

Reviewer 2 Report

The article is well-written and the authors have covered the interesting subject in a succinct manner.

While the manuscript has significant scientific merit there is a number of awkward expressions in the manuscript (e.g. "The disappeared rates of SVOs..") and errors in grammar (.."the resluts") and use of English in general that need to be looked through with the aid of a native speaker or a proofing service.

Methods - Results

The authors mention only the use of a paired-samples t-test for before and after results among the groups but do not mention the statistical test (if any) to test between the placebo and dose-dependent groups. Was there such a test? It appears there was none. I would suggest a mixed model design to test the between-group differences so as to ascertain the dose-dependent improvement in those who received the enzymes in question.

Discussion

The discussion section is generally well-written but the final version needs to take into account the results from the between-groups comparison (see above).

Also the reference style employed in the manuscript does not correspond to the appropriate reference style. Please amend.

 

Author Response

Dear reviewer,

 

Thank you for the precious suggestions on the manuscript, “Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin Supplement,” and we have made substantial revisions to the manuscript based on the reviewer’ comments, as the details below.

1.The article is well-written and the authors have covered the interesting subject in a succinct manner.While the manuscript has significant scientific merit there is a number of awkward expressions in the manuscript (e.g. "The disappeared rates of SVOs..") and errors in grammar (.."the resluts") and use of English in general that need to be looked through with the aid of a native speaker or a proofing service.

Methods – Results The authors mention only the use of a paired-samples t-test for before and after results among the groups but do not mention the statistical test (if any) to test between the placebo and dose-dependent groups. Was there such a test? It appears there was none. I would suggest a mixed model design to test the between-group differences so as to ascertain the dose-dependent improvement in those who received the enzymes in question.

Discussion The discussion section is generally well-written but the final version needs to take into account the results from the between-groups comparison (see above). Also the reference style employed in the manuscript does not correspond to the appropriate reference style. Please amend.

Response: (1)The manuscript has been rewritten, and edited by a native English speaker.

(2) Furthermore, a statistical test was performed to compare between the dose-dependent and placebo groups by Williams’ test. The results show that all of the percentages of disappeared rate in LPG, MPG and HPG are statistically significant when compared with placebo group (Table 2). Ocular examinations in this study found that the SVOs had been absorbed in the “successful” cases, thus, our capsules should enhance the functions of hydrolysis and lysis. Furthermore, we also found that SOVs from patients with diabetic retinopathy could be dissolved by fruit enzymes, and the mechanism of disappearance of VH may be primarily due to the anti-thrombotic function of papain. Therefore, we suggest that a combination of bromelain, papain, and ficin can easily dissolve and absorb ocular opacities.

    An ocular floater or opacity is the result of opaque fragments floating in the vitreous. This study shows that most of the ocular floaters did not impact vision; and the numbers of floaters sometimes increased with age. Although the symptoms were minimal, they caused significant impairment in vision-related quality of life for some victims﹝24﹞. While the SOVs may occur suddenly and bother the subjects, patients might adapt to vitreous opacities over a 3-month period. SVOs can be associated with PVD with or without retinal breaks. Moreover, some ocular floaters may be caused by the solitary blood clots of various retinopathies, such as diabetic retinopathy and intraocular blood due to trauma.

                                                                                                    

Table 1: The changes in various floaters before and after taking the fruit protease supplements over a 3-month period

Time Number	Baseline	1st month	2nd month	3rd month
One   Multiple	80   40	78      37	68       30	24* (30.0%)   11* (27.5%)

   N= 190 eyes

1. Multiple floaters mean a patient with at least 2 ocular floaters in the visual field.
2. After treatment, multiple floaters may completely or incompletely disappear; therefore, multiple floaters would change to 1 or 0 floaters.
3. All 190 patients took one capsule twice daily, and every fruit protease capsule included 190 mg bromelain, 95 mg papain, and 95 mg ficin.

*4. The results at the baseline and after 3 months were compared by paired t-test, where a p value less than .05 means significant difference.

 

Table 2: The changes in ocular floaters before and after the fruit protease supplements 

over a 3-month period

Time Capsule daily	Baseline	1st month	2nd month	3rd month
none   1 capsule   2 capsules   3 capsules	40       40      40      40	40      38      35      34	40         33       32       30	38 (95.0%)   14* (35.0%)#   12* (30.0%)#   10* (25.0% )#

1. Group 1 (N= 40 eyes): no protease supplements were taken (placebo group). Group 2 (N= 40 eyes): all patients took 1 mixed protease capsule including 190 mg bromelain, 95 mg papain, and 95 mg ficin every day. Group 3 (N= 40 eyes): all patients took 2 capsules daily. Group 4 (N= 40 eyes): all patients took 3 capsules daily.

*2. The results at the baseline and after 3 months were compared by pair-t test, where a p value less than .05 means a significant difference.

^#3. We compared the results between the placebo and dose-dependent groups after 3-month therapies by Williams’ test, where a p value less than .05 means significant difference.

 

    Ocular floaters may be bothersome during the daily life of victims. For example, Vanova et al. demonstrated that floaters could impact the quality of life with respect to vision-dependent tasks, and found that more than two-thirds of the patients had moderate to extreme difficulty in reading small print and driving at night﹝25﹞. Chronic floaters may be just as problematic for some individuals as other well-established ocular conditions that are viewed as having more impact on visual functioning, such as cataracts or macular membranes. Additionally, Wagle et al. concluded that degenerative SVOs had negative impact on patient’s health-related life﹝24﹞. On average, the cohort studied by this group was willing to accept a 7% risk of blindness to eliminate SVOs, and this risk is comparable to the associated risk of treating diabetic retinopathy and age-related macular degeneration. Obviously, the safe use of new methods for treating SVOs is important.

 

With above responses and substantial revisions, please kindly consider its publication in Polymers. Thank you for your kind consideration and assistance.

 

Sincerely,

 

Chi-Ting Horng M.D., Ph.D.

Mail: [email protected]

 

Author Response File: Author Response.docx

Reviewer 3 Report

The paper presented for peer-review presents the results of screening studies on the effects of several plant enzymes found in fruit juices on Symptomatic Vitreous Opacities of eyes. I don't know why the results of this type of research were sent to Polymers. This is probably a some mistake. By no means these studies fall under the topic of this journal. For this reason, they cannot be published here.

Author Response

Dear reviewer,

Thank you for the precious suggestions on the manuscript, “Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin Supplement,” and we have made substantial revisions to the manuscript based on the reviewer’ comments, as the details below.

 

1. The paper presented for peer-review presents the results of screening studies on the effects of several plant enzymes found in fruit juices on Symptomatic Vitreous Opacities of eyes. I don't know why the results of this type of research were sent to Polymers. This is probably a some mistake. By no means these studies fall under the topic of this journal. For this reason, they cannot be published here.

Response: Collagen fibers and hyaluronic acid in the vitreous of human eyes belongs to advances in optical polymers applied in the specific chemical environment.

 

With above responses and substantial revisions, please kindly consider its publication in Polymers. Thank you for your kind consideration and assistance.

 

Sincerely,

 

Chi-Ting Horng M.D., Ph.D.

Mail: [email protected]

 

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The Manuscript authored by Takeuchi entitled "Treatment of Symptomatic Vitreous Opacities with Pharmacologic Vitreolysis by Using a New Method of a Mixed Bromelain, Papain and Ficin" was modified and re-submitted to revision.

 

Authors answered some queries but as mentioned before, it still lacks evidences. Besides, Polymers is not the proper journal for this kind of research. There are no polymer chemistry or innovation on polymer sciences. I recommend other MPDI Journals instead, such as Pharmaceuticals (ISSN 1424-8247) or Pharmaceutics (ISSN 1999-4923), which are more appropriate for these clinical results.

This manuscript still needs to clarify about bioavailability of these commercial enzymes.

As reviewed before and answers were not enough to clarify:

(i) How authors can guarantee that the enzymes would stay integer in the digestive system and gastrointestinal tract. Also, no evidences were showed demonstrating that the enzymes would be absorbed and reach vitreous liquid inside human eyes. And about ADME and the first pass effect?

(ii) I recommend extending some experiments by determining availability and absorption of these enzymes.

In this format, it should not be considered for publication in Polymers. It does not fit with Polymers aims and scope.

Reviewer 3 Report

 Reading Scope Polymers posted on the website https://www.mdpi.com/journal/polymers/about it is clear that the work presented by the authors could only possibly be the subject of the section:

Polymer Application (biomedical application of polymeric materials)

Unfortunately, the work did not present the construction or modification of polymeric materials, or their influence on the treatment process (such types of works are included in this section). The subject of this manuscript is only the results obtained during screening tests carried out on a group of patients with vision disease who have been administered certain enzymes and the morphological changes in the eyes (changes in the structure of certain proteins) have been examined. And that's it, it has nothing to do with even the most widely understood science and research on the application of polymers in medicine. It certainly does not fit in the  presented thematic scope of "Polymers". All conclusions of the authors regarding changes in the structure of proteins are speculative, not supported by any specific evidence (no basic studies on the composition and morphology of these proteins) I strongly advise against accepting this manuscript for printing in Polymers. The presented studies are typically medical studies.