Insulin Nanoemulsion Eye Drops for the Treatment of Dry Eye Disease in Sjögren’s Disease: A Randomized Clinical Trial Phase I/II

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

I agree that this manuscript can be published. Congratulation.

Author Response

Comment: I agree that this manuscript can be published. Congratulation.

R: Thank you very much for your time and observations. 

Reviewer 2 Report

Comments and Suggestions for Authors

The article is very well structured and presents an important study for the treatment of DED. However, some considerations should be observed.

 

There are so many old references and  some are older than 5 years. It is necessary to update. It will help to prove the sequence of studies and the current relevance of the results obtained in this research. The introduction has 16 of the 26 references cited, older than 5 years and some older than 10 years. Provide a justification in the text, if the old reference is important for the chronology of research on DED.

Although it is not the focus of the study, the characteristics and studies on the insulin emulsification process were not addressed. In the methodology, insulin was added at the end of the process, leaving room for questioning whether it was correctly dispersed and nanoparticulated. The particulation is essential to increase bioavailability. Scientific data can be used to better explain this part of the processing and preparation of the nanoemulsion. Understanding this process is essential to understanding the results, since there was no significant difference between treatment and placebo, which may be linked to the bioavailability of insulin.

Author Response

Comments:

1. The article is very well structured and presents an important study for the treatment of DED. However, some considerations should be observed.

Reply: Thank you

2. There are so many old references and  some are older than 5 years. It is necessary to update. It will help to prove the sequence of studies and the current relevance of the results obtained in this research. The introduction has 16 of the 26 references cited, older than 5 years and some older than 10 years. Provide a justification in the text if the old reference is important for the chronology of research on DED.

Reply: Thank you for the observation. The reason for mentioning those old references is to present the concepts that support the mechanisms of action of the insulin hormone in the eye. Several clinical series have been published that encourage the study in recent years. However, most human, experimental, and basic science studies addressing the mechanism of action were published in the medical literature 10 years ago or before.  They provide the background and justification for the clinical research. Since the whole study does not bring excessive references (42 in this revised version),  we believe that references help understand the study. 

3. Although it is not the focus of the study, the characteristics and studies on the insulin emulsification process were not addressed. In the methodology, insulin was added at the end of the process, leaving room for questioning whether it was correctly dispersed and nanoparticulated. The particulation is essential to increase bioavailability. Scientific data can be used to better explain this part of the processing and preparation of the nanoemulsion. Understanding this process is essential to understanding the results, since there was no significant difference between treatment and placebo, which may be linked to the bioavailability of insulin.

Reply: 

We appreciate the reviewer's observation and the opportunity to clarify this aspect in more detail. Although insulin is a hydrophilic protein and was added after the emulsification step, several studies have demonstrated that nanoemulsions can effectively incorporate and stabilize therapeutic proteins and peptides post-emulsification, provided that appropriate surfactants are used (Tsai et al., 2014 - DOI:10.1371/journal.pone.0102850; Ali et al., 2022 - DOI: 10.1016/j.jddst.2022.103338). Our nanoemulsion, currently under patent application, contains medium-chain triglycerides stabilized by a combination of non-ionic surfactants, phospholipids, and a cationic co-surfactant that also acts as a preservative. These components promote interfacial stabilization and facilitate favorable interactions with the ocular surface, enhancing mucoadhesion and residence time, key features for improving bioavailability in topical ocular delivery. Moreover, nanoemulsions are known to restore the lipid layer of the tear film, which is directly relevant for dry eye disease (Chatzidaki & Mitsou, 2025 - 10.3390/pharmaceutics17030337). This intrinsic therapeutic action may partially explain the absence of a significant difference between the insulin and placebo groups since both received the exact nanoemulsion vehicle.
Both insulin-loaded and placebo nanoemulsions were characterized by dynamic light scattering to determine mean droplet size, polydispersity index, and zeta potential. Osmolarity and pH were also evaluated to ensure the formulations' suitability for ocular administration. No significant differences were observed between the two formulations in any of these parameters, confirming that the incorporation of insulin did not affect the nanoemulsion's colloidal stability or physicochemical properties. The detailed characterization results have now been included in the revised version of the manuscript.
It is also important to note that the aqueous phase of a nanoemulsion provides a microstructured environment distinct from simple aqueous solutions due to the presence of surfactants and nanoscopic interfaces. This environment promotes protein dispersion and stability and improves retention and spreading over the ocular surface.
These clarifications and references have been included in the revised manuscript (attached here as Manuscript Vision 3631721 R1.pdf) to contextualize the formulation strategy better and to explain the potential limitations related to bioavailability and the observed therapeutic outcomes.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the scientific work- It is an important information for dry eye treatment.

The main question is whether insulin eye drops improve dry eye signs and symptoms. Until now literature is very good for insulin and corneal epithelial defects, but not for dry eye therapy.

It is a homogen study population (patients with severe dry eye syndrome) which was splitted in two groups  one with insulin eye drops, the second with the same solution but without insulin.subjective symptoms and opjective dry eye signs  are evaluated

 

Author Response

Comments:

1. Thank you for the scientific work- It is an important information for dry eye treatment.

Response: Thank you for the kind comment. 

2. The main question is whether insulin eye drops improve dry eye signs and symptoms. Until now literature is very good for insulin and corneal epithelial defects, but not for dry eye therapy.

Response: Our study detected that insulin for 30 days is safe in Sjogren´s disease patients. However, the study did not find elements of efficacy. The potential limitations are presented in the discussion and conclusions. Novel studies will check the variables to confirm the lack of efficacy or to identify the ideal formula and indications. 

3. It is a homogen study population (patients with severe dry eye syndrome) which was splitted in two groups  one with insulin eye drops, the second with the same solution but without insulin.subjective symptoms and opjective dry eye signs  are evaluated.

Response: Thank you for your observations. We agree that the control group benefited from the vehicle solution. Further studies with more sensitive tools will check this information. 

Reviewer 4 Report

Comments and Suggestions for Authors

As a peer reviewer, I recommend rejection of the manuscript titled "Insulin nanoemulsion eye drops for the treatment of dry eye disease in Sjögren’s Disease: A randomized clinical trial phase I/II" based on the following major and minor issues:

 

Major Reasons for Rejection:

1. Lack of Statistically Significant Efficacy

• The primary and secondary outcomes (Schirmer I test, OSDI, TBUT, CF, LG) showed no statistically significant difference between insulin and placebo groups (p > 0.05).
• While some improvements were observed, they occurred in both arms, strongly suggesting a placebo effect or variability over time rather than any insulin-specific benefit.
• The authors themselves admit that “clinical trials, including this one, have not demonstrated the efficacy of insulin eye drops for treating DED” (line 266).

2. Small Sample Size and High Dropout Rate

• Only 23 participants completed the trial (10 insulin, 13 placebo), from an initial 99 considered. This is underpowered for detecting subtle therapeutic effects.
• Nearly 30% dropout, and very low adherence in the placebo group (15.38%) further undermines internal validity.

3. Methodological and Design Weaknesses

• Single-center, non-stratified trial with an inadequate sample to represent the clinical variability in Sjögren’s disease-related DED.
• No power calculation presented to justify sample size.
• Short duration (4 weeks) for a chronic disease like DED is inadequate to assess true clinical efficacy.
• Insufficient details are given about randomization process, blinding validation, and compliance assurance.

4. Conflict of Interest

• Two lead authors hold patents on the insulin nanoemulsion technology under investigation.
• While disclosed, this introduces bias risk, especially in interpreting ambiguous findings as positive or suggesting further trials without demonstrable benefit here.

5. Inconclusive Interpretation

• Despite negative results, the discussion remains optimistic and speculative (“positive trend,” “safe,” “encouraging comfort”), which conflicts with the data and undermines scientific rigor.
• The manuscript attempts to justify null results by discussing protocol limitations but fails to propose a concrete, data-driven way forward.
• Writing clarity: Some parts of the manuscript contain long, meandering sentences that obscure meaning. For example, sections of the discussion are repetitive and speculative.

• Presentation: Tables are dense and poorly integrated into the narrative.
• Statistical transparency: The manuscript lacks effect sizes, confidence intervals, and power analysis that are critical for clinical interpretation.

 

Conclusion:

The concept of insulin nanoemulsions for DED is intriguing and deserves future exploration. However, this particular clinical trial does not meet the scientific or methodological standards necessary to support publication in a peer-reviewed journal. It fails to demonstrate efficacy, has severe limitations in trial design, and presents an overly optimistic narrative inconsistent with the data. I therefore recommend rejection in its current form.

Author Response

Comment:

1. Lack of Statistically Significant Efficacy

• The primary and secondary outcomes (Schirmer I test, OSDI, TBUT, CF, LG) showed no statistically significant difference between insulin and placebo groups (p > 0.05).
• While some improvements were observed, they occurred in both arms, strongly suggesting a placebo effect or variability over time rather than any insulin-specific benefit.
• The authors themselves admit that “clinical trials, including this one, have not demonstrated the efficacy of insulin eye drops for treating DED” (line 266).

Response: Thank you for your observation. Due to the lack of prior randomized clinical trials addressing the issue, we believe that results, even with a lack of statistically significant efficacy, are a relevant study because they can support decision-making and future clinical trial design regarding the safety of the insulin nanoemulsion eyedrops and the formulation dose studied.

2. Small Sample Size and High Dropout Rate

• Only 23 participants completed the trial (10 insulin, 13 placebo), from an initial 99 considered. This is underpowered for detecting subtle therapeutic effects.
• Nearly 30% dropout, and very low adherence in the placebo group (15.38%) further undermines internal validity.

Response: Thank you for this relevant criticism. The small sample size and high dropout rate were caused in part by the study conducted during the pandemic. Patients were infected, were suspicious of COVID-19 infection, or were afraid to attend the hospital appointment due to the risk to themselves or their families. Showing this limitation in the discussion session and measuring the rate of protocol adherence by the volunteers is valuable information to the reader and supports future decisions on this topic and other therapeutic clinical trials involving eye drops. 

3. Methodological and Design Weaknesses

• Single-center, non-stratified trial with an inadequate sample to represent the clinical variability in Sjögren’s disease-related DED.
• No power calculation presented to justify sample size.
• Short duration (4 weeks) for a chronic disease like DED is inadequate to assess true clinical efficacy.
• Insufficient details are given about randomization process, blinding validation, and compliance assurance.

Response:  Thank you for the valuable observations. Our option for a single-center study will be considered in future studies on dry eye disease. SjD was chosen to avoid the clinical variability regarding the many causes of dry eye; however, we agree that we observed variability even in a single disease, such as SjD. We did the power calculation and aimed to have at least 15 individuals in each group. However, this study revealed a dropout in both arms and a lack of minimal compliance. These observations of dropout and lack of compliance are present in the text and will be helpful for future studies in dry eye treatment. The suggestion for a more extended period of study will also be considered. 

The process of randomization and blind validation is described in the paragraph starting on page 2, line 76, and was strictly observed by the other research group. The compliance assurance was observed, and the rules and guidelines for clinical research of the Declaration of Helsinki, the University of Sao Paulo, and the Brazilian Ethical Committee of Ethics I Research were previously approved and observed by the research group in all the study steps (mentioned on page 2, line 68).  

4. Conflict of Interest

• Two lead authors hold patents on the insulin nanoemulsion technology under investigation.
• While disclosed, this introduces bias risk, especially in interpreting ambiguous findings as positive or suggesting further trials without demonstrable benefit here.

Response: We agree that researchers and inventors are at risk for their ideas and products. We believe publishing the results of studies and allowing other researchers to observe the limitations and make critical improvements will induce significant advances in medical science and population health. 

5. Inconclusive Interpretation

• 1. Despite negative results, the discussion remains optimistic and speculative (“positive trend,” “safe,” “encouraging comfort”), which conflicts with the data and undermines scientific rigor.

Response: We respectfully disagree that our discussion session conflicts with the data and undermines scientific rigor. After 102 human studies addressing insulin eyedrops in human eyes in PubMed, none studied adherence, safety, or other dry eye diseases other than diabetes mellitus in a randomized trial. Our discussion mentions the results and indicates their limitations (page 9, line 249). 

• 2. The manuscript attempts to justify null results by discussing protocol limitations but fails to propose a concrete, data-driven way forward.

Response: We would like to thank the reviewer for her/his comment. On page 10, line 286 of the discussion, we emphasized that no clinical trial detected clinical benefits for dry eye using insulin eyedrops, despite benefits in some biomarkers detected in tears. Suggestions for future trials were mentioned in the sequence in the same paragraph. 

3. Writing clarity: Some parts of the manuscript contain long, meandering sentences that obscure meaning. For example, sections of the discussion are repetitive and speculative. 

• Presentation: Tables are dense and poorly integrated into the narrative.
• Statistical transparency: The manuscript lacks effect sizes, confidence intervals, and power analysis that are critical for clinical interpretation.

Response: We would like to thank the reviewer for the comments. These subjective observations are critical to improving our manuscripts, and on some points, we respectfully disagree. A broad discussion will be helpful for future researchers in the field. Our tables were carefully designed to support the reading, and the confidence intervals were presented in Table 3. The statistical analysis is described in detail in item 2.5, page 4.  The aspects mentioned above harmed the effect sizes and power analysis (item 3). 

 

Conclusion:

The concept of insulin nanoemulsions for DED is intriguing and deserves future exploration. However, this particular clinical trial does not meet the scientific or methodological standards necessary to support publication in a peer-reviewed journal. It fails to demonstrate efficacy, has severe limitations in trial design, and presents an overly optimistic narrative inconsistent with the data. I therefore recommend rejection in its current form.

Response: Considering the relevance of the topic, the transparency applied to report the study with insulin eyedrops, and the utility for clinical trials with limitations to support better future study design, we hope the reviewer reconsider her/his decision and allow the publication of this manuscript. 

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

Manuscript Ready For publication