Assessing the Effectiveness of 0.01 percent HOCL Interventions in Managing Ocular Surface Disorders: Randomized Trials on Dry Eye Disease and Meibomian Gland Dysfunction

Dry eye disease and meibomian gland dysfunction (mgd) represent two prevalent ocular surface disorders that significantly impact individuals’ quality of life and ocular health. Understanding the complexities of these conditions and their treatment challenges is crucial for clinicians and researchers alike. Dry eye disease is characterized by inadequate tear production or poor tear quality leading to ocular discomfort visual disturbances and potential damage to the ocular surface. Meibomian glands which secrete the lipid component of tears play a crucial role in maintaining tear film stability dysfunction of these glands known as meibomian gland dysfunction (MGD). MGD results in altered tear composition and instability contributing to evaporative dry eye. One of the primary challenges in treating dry eye and mgd lies in their multifactorial nature. These conditions can arise from various etiologies including age related changes hormonal imbalances, environmental factors and systemic diseases. Consequently, developing a tailored treatment approach that addresses individual patient needs and underlying causes can be complex. Traditional treatment options for dry eye and mgd include artificial tears, lubricating eye drops and warm compresses. While these interventions can provide symptomatic relief for some patients, they often fail to address the underlying pathophysiology of the conditions. Moreover frequent application of lubricating eye drops may wash away natural tear components and exacerbate symptoms. Over time in recent years there has been growing interest in novel therapeutic approaches for managing dry eye and mgd. Lipid based formulations such as lipid emulsions and liposomal sprays aim to restore the lipid layer of the tear film and improve tear stability, however the efficacy of these formulations varies among individuals and long term outcomes remain unclear. Another promising avenue of research involves the use of anti inflammatory agents to target underlying inflammation associated with dry eye and mgd. Topical corticosteroids and immunomodulatory drugs have shown efficacy in reducing ocular surface inflammation and improving symptoms in some patients. However concerns regarding potential side effects including intraocular pressure elevation and cataract formation limit their long term use. Furthermore there is a lack of consensus regarding standardized diagnostic criteria and outcome measures for assessing dry eye and mgd severity and treatment response. Objective diagnostic tools such as tear film osmolarity measurement and meibography imaging offer valuable insights into ocular surface health but may not always correlate with patients symptoms or treatment outcomes. Moreover, patient adherence to prescribed treatments poses a significant challenge in managing dry eye and mgd effectively. The need for frequent application of eye drops or adherence to daily warm compress regimens can be burdensome for patients leading to treatment discontinuation or suboptimal outcomes in conclusion dry eye disease and meibomian gland dysfunction. Present complex challenges in diagnosis and treatment despite advances in our understanding of these conditions there remains a need for targeted therapies that address underlying pathophysiological mechanisms while minimizing treatment burden and side effects. Hence, further avenues need to be studied to help this prevalent condition amongst our population.

 1.     Rationale of the study (up to 500 words): Mention how the research question addresses the critical barrier(s) in scientific knowledge, technical capability, and/or programmatic/ clinical/lab practice and its relevance to local, national and international context with relevant bibliography.

HOCL has multifaceted applications in dermatology, wound healing, eye care, and dentistry. It is the ordinary disinfectant in industrial domestic and medical aspects and has the same active components of household bleach but with a different chemical composition. Impurity-free HOCl originated from the products of the human immune response. Across the oxidative reaction, highly activated molecules such as HOCl are stimulated from the leukocyte’s action to external microorganisms. Due to its quickly neutralized feature, HOCL is nontoxic to the ocular surface. HOCl, a major inorganic bactericidal compound of innate immunity, is naturally produced as part of the cytotoxic myeloperoxidase system in neutrophils.1In vitro, against microorganisms, HOCl causes oxidation of nucleotides, inactivation of cell enzymes, disruption of cell membranes and rapid cell lysis.2 Consequently, solutions containing HOCl are highly active against all bacterial, viral and fungal human pathogens. Based on in vitro studies, antimicrobial activity of 0.01% HOCl appears to be superior to other commonly used skin antiseptics but without cytotoxicity. Moreover, HOCl was well tolerated after continuous use and was not toxic to the human ocular surface.3-7 Consequently, these characteristics make HOCl an ideal adjuvant treatment for managing eye infections.8,9  Use of HOCl ophthalmic spray is indicated as an adjuvant in treating blepharitis and for cleansing the periocular area in the days before or after ocular surgery.10,11 Although bacteria may or may not initiate blepharitis, the oily and inflamed eyelid margins of patients with blepharitis are frequently colonized by bacteria. Hence, a proper assessment of HOCL as eye drop and eyelid wipe in improving dry eye symptoms and reducing MGD symptoms needs to be studied through a randomized controlled trial.

 References:

1.     Wang L, Bassiri M, Najafi R, et al. Hypochlorous acid as a potential wound care agent: part I. Stabilized hypochlorous acid: a component of the inorganic armamentarium of innate immunity. *J Burns Wounds.*2007;6:e5.

2.     Sakarya S, Gunay N, Karakulak M, et al. Hypochlorous acid: an ideal wound care agent with powerful microbicidal, antibiofilm, and wound healing potency. *Wounds.*2014;26(12):342–350.

3.     Li Z, Wang H, Liang M, et al. Hypochlorous acid can be the novel option for the meibomian gland dysfunction dry eye through ultrasonic atomization. *Dis Markers.*2022;2022:8631038.

4.     Fam A, Finger PT, Tomar AS, Garg G, Chin KJ. Hypochlorous acid antiseptic washout improves patient comfort after intravitreal injection: a patient reported outcomes study. *Indian J Ophthalmol.*2020;68(11):2439–2444.

5.     Anagnostopoulos AG, Rong A, Miller D, et al. 0.01% hypochlorous acid as an alternative skin antiseptic: an in vitro comparison. *Dermatol Surg.*2018;44(12):1489–1493.

6.     Armstrong DG, Bohn G, Glat P, et al. Expert recommendations for the use of hypochlorous solution: science and clinical application. *Ostomy Wound Manage.*2015;61(5):S2–S19.

7.     Finger PT, Fam A, Tomar AS, Garg G, Chin KJ. COVID-19 prophylaxis in ophthalmology. *Indian J Ophthalmol.*2020;68(10):2062–2063.

8.     Harsch AG, Stout N, Lighthizer N. Beat the blepharitis blues. *Rev Cornea Contact Lens.*2016;153(7):12–15.

9.      Kern JR, Fahmy AH. Dry eye patients report improvement in symptoms with hypochlorous acid use over 30 days. *Invest Ophthalmol Vis Sci.*2019;60(9):6740.

10. Ocudoxâ„¢ Summary of Product Characteristics. https://www.alfaintes.it/it/prodotto/18 .

11. Stroman DW, Mintun K, Epstein AB, et al. Reduction in bacterial load using hypochlorous acid hygiene solution on ocular skin. *Clin Ophthalmol.*2017;11:707–714.

  2.     Hypothesis/ Research question (up to 100 words): Please provide details in PICO format as applicable.

 Patient- Cases of dry eye disease who are unable to get relief with the current treatment.

**Intervention-**0.01% HOCL eye drops for dry eye patients and 0.01% HOCl eyelid wipes for mgd cases

 Control- Dry eye cases with conservative treatment (lubricant eye drops)

 Outcome- symptomatic relief from dry eye symptoms. Reduction in the level of ocular surface immune cell phenotype.

  **3.****Study Objectives (**Define the objectives clearly and in measurable terms; mention as primary and secondary objectives, if necessary. Do not include more than 3-4 objectives. Provide specific, measurable and timebound outcomes for each objective.)

Primary objectives:

**1.     1.**Evaluate the efficacy of 0.01% HOCL interventions in alleviating symptoms and improving ocular surface health in individuals with dry eye disease.

**2. 2.**Investigate the effectiveness of 0.01% HOCL eyelid wipes/spray in managing symptoms and addressing ocular surface abnormalities associated with Meibomian Gland Dysfunction (MGD).

Secondary objective:

**1.**To understand the effect of 0.01% HOCL drops in (Neutrophil extracellular traps) NETs and ocular surface immune cell phenotype in dry eye and MGD.

Primary objective 1: Evaluate the efficacy of 0.01% HOCL interventions in alleviating symptoms and improving ocular surface health in individuals with dry eye disease.**

a.     Study design

This will be a randomized control prospective trial. There will be two arms to this study

**Intervention Arm 1: Conservative treatment (CT)

• 0.01% HOCL (hypochlorous acid) eye drops**

Control Arm 2: Conservative treatment only  (lubricants)

 b.     Study area (multiple choice): Community/Hospital/Laboratory

All clinically diagnosed cases of Dry eye reporting at the outpatient department of Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India will be screened as per the study inclusion/exclusion criteria after patient consent for the study.

Inclusion criteria:

1.     Patient age between 18-70 years

2.     Cases with no history of prior ocular surgery for past six month.

Exclusion criteria:

1.     Patient age below 18 years

2.     Pregnant or lactating females

3.     Any prior ocular surgery in past six months

4.     Those with ocular problems such as ocular allergy, and nasolacrimal sac problems; those who are using a punctual plug or contact lenses

Ethics Review:

1.     The study will be initiated after obtaining ethical approval. Ethical approval will be taken from the Institutional ethics committee of All India Institute of Medical Sciences, New Delhi.

2.     Since this study also included use of an interventional drug, a CTRI approval will also be obtained prior to study initiation.

3.     As part of the institutional ethics, informed consent having details on study risks and benefits will be obtained from the participating patients during enrolment.

 c.     Sample size estimation and sampling strategy

There was not a previous study that has directly related studied the subjective complains and objective data with topical 0.01% HOCL drops in dry eye. The arbitrary effect size 0.25 was taken with power of study 90% (critical factor F=3.89, non-centrality parameter λ= 10.687) was selected to count the minimum sample for this study and was estimated to be 171.  In view of this, we will be recruiting 90 patients (180 eyes) in intervention arm a****nd 90 patients (180 eyes) in control arm

d.     Project implementation plan

Participating departments- The study will be conducted in Dr. R.P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi

Clinical work up and investigations:

a.     Dry Eye Diagnosis and Classification

Subjective symptoms will be graded on a serial scale from 0 to 4, according to the verified 12-item ocular surface disease index (OSDI) questionnaire. The total Ocular Surface Disease Index (OSDI) marks range from 0 to 100 and was calculated using the following equation: OSDI=the summary of scores for every question answered × 100)/(overall number of answered questions × 4).

The standards for the diagnosis of dry eye are as follows:

1.     Ocular surface disease index (OSDI) score of less than 12

2.     Without tear film outliers (tear film break-up time, TBUT > 5 seconds, and Schirmer’s test value of >5 mm after 5 min)

3.     Lack of evidence of corneal or conjunctive epithelial erosion with fluorescent staining

4.     Normal lid margins or meibum

Description of Intervention:

Group 1 -Interventional agent: 0.01% Hypochlorous acid (ph-6.5) in Nacl sterile solution administered four times a day for a treatment period of three month.

Group 2- Comparator agent: preservative free lubricant (carboxy methyl cellulose drops) will be administered to four times a day.

Clinical Investigations in the Study:

The following investigations will be performed at the Day 1, Day 15th, Day 30th, Day 60th, Day 90th.

1.     Best Corrected Visual acuity (BCVA) to document the visual acuity.

2.     Schirmer’s test to evaluate the tear secreted.

3.     Tear-film Break Up time (TBUT) to see the status of tear film stability

4.     Clinical photograph before, during and after study

IDRA ocular surface analyzer will be used for evaluating the following parameters for each patient

1. Non-invasive tear film break-up time

2. Tear meniscus height

3. Lipid layer interferometry

4. Eye blink quality

5. Infrared meibography to assess meibum quality, and grade of meibomian gland dysfunction

6. In vivo confocal microscopy (IVCM) to assess the corneal nerves and its damage in dry eye

 

Clinical
parameters

Follow up & investigation timepoints

|Day 0

Day 15

Day 30

Day 60

Day 90

|Visual acuity (BCVA)

X

x

x

x

x

|Schirmers test (mm)

X

x

x

x

x

|TBUT (sec)

X

x

    |I

D

R

A

  Tear Meniscus

Height

X

x

x

x

x

|Lipid Layer Interferometry

X

x

x

x

x

|Eye Blink Quality

X

x

x

x

x

|Infrared Meibography

X

x

x

x

x

|IVCM

X

x

x

x

x

|Inflammadry MMP9

X

x

x

x

x

X : will be done

Adverse events

Monitoring of adverse events and ocular discomfort will be done at each follow up and appropriate measure will be taken by the clinician.

e.     Design of statistical analysis

e.

The findings will be reported as Mean ± Standard error of Mean (SEM) and represented as bar graphs. Differences in the various parameters between the groups will be tested for statistical significance by Mann–Whitney test using GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA). Correlation among the various study parameters will be determined by Spearman Rank correlation tests using MedCalc® Version 12.5 (MedCalc Software, Ostend, Belgium). P < 0.05 will be considered to be statistically significant.

  

Clinical
parameters

Follow up & investigation timepoints

Statistical tests to be done

|Day 0

Day 15

Day 30

Day 60

Day 90

|Visual acuity (BCVA)

x

x

x

x

x

  Chi2 test/

Fisher’s exact test

|Schirmers test (mm)

x

x

x

x

x

|TBUT (sec)

x

x

    |I

D

R

A

  Tear Meniscus

Height

x

x

x

x

x

|Lipid Layer Interferometry

x

x

x

x

x

|Eye Blink Quality

x

x

x

x

x

|Infrared Meibography

x

x

x

x

x

|IVCM

x

x

x

x

x

|Inflammadry MMP9

x

x

x

x

x

 Primary objective 2: Investigate the effectiveness of 0.01% HOCL eyelid wipes/spray in managing symptoms and addressing ocular surface abnormalities associated with Meibomian Gland Dysfunction (MGD).

a.     Study design

This will be a randomized control prospective trial. There will be two arms to this study

Intervention Arm 1: Conservative treatment (CT) + 0.01% HOCL (Hypochlorous acid in Nacl) sterile eyelid wipes

Control Arm 2: Conservative treatment only (lubricant)

 b.     Study area (multiple choice): Community/Hospital~~/Laboratory~~

 All clinically diagnosed cases of Meibomian gland dysfunction reporting at the outpatient department of Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi, India will be screened as per the study inclusion/exclusion criteria after patient consent for the study.

Inclusion criteria:

1.     Patient age between 18-70 years

2.     Cases with no history of prior ocular surgery for past six months

Exclusion criteria:

3.     Patient age below 18 years

4.     Pregnant or lactating females

5.     Any prior ocular surgery in past six months

6.     Those with ocular problems such as ocular allergy, and nasolacrimal sac problems; those who are using a punctual plug or contact lenses

 Ethics Review:

1.     The study will be initiated after obtaining ethical approval. Ethical approval will be taken from the Institutional ethics committee of All India Institute of Medical Sciences, New Delhi.

2.     Since this study also included use of an interventional drug, a CTRI approval will also be obtained prior to study initiation.

3.     As part of the institutional ethics, informed consent having details on study risks and benefits will be obtained from the participating patients during enrolment.

  c.     Sample size estimation and sampling strategy

 There was not a previous study that has directly related studied the subjective complains and objective data with topical 0.01% HOCL drops in dry eye. The arbitrary effect size 0.25 was taken with power of study 90% (critical factor F=3.89, non-centrality parameter λ= 10.687) was selected to count the minimum sample for this study and was estimated to be 171.

In view of this, we will be recruiting 90 patients (180 eyes) in intervention arm and 90 patients (180 eyes) in control arm

 f.      Project implementation plan

 Participating departments- The study will be conducted in Dr. R.P. Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi.

 The participating departments in the study are:

*a.*Department of Ophthalmology – The Principal Investigator (Dr. Namrata Sharma) and the co- investigator (Dr. Prafulla Maharana) will help in enrolling patients in the outpatient department.

Clinical work up and investigations:

1.     Patient recruitment

 The criteria for the MGD group are as follows:

-Presence of symptoms (OSDI ≥ 12)

-At least one lid margin abnormality

-Poor meibomian gland expression (grade ≥ 1) or worse qualitative variety in meibum (meibum quality score ≥ 3).

The eyelid margins and meibomian glands will be examined for lid margin anomalous, gland expression, and meibum amount and colour. Lid margin anomalous will be scored as 0 (absent) or 1 (present) for the following parameters: plugged meibomian gland orifices, vascular congestion, irregularity of the lid margin, and partly expressions of the mucocutaneous borderline. The extent of meibomian gland expression, using steady digital pressure applied on five glands of the middle third of the lower lid, will be graded as such: grade 0, all five glands expressible; grade 1, three or four glands expressible; grade 2, one or two glands expressible; and grade 3, no glands expressible. The meibum attribute on eight lower lid glands will be graded as follows: grade 0, clear; grade 1, cloudy; grade 2, cloudy with granular particulates; and grade 3, thick, like toothpaste-like particulates. Each of the eight glands of the lower eyelid will be graded on the scale from 0 to 3.

Description of Intervention:

Group 1 -Interventional agent: 0.01% Hypochlorous acid (ph-6.5) in Nacl sterile wipes administered three times a day for a treatment period of three month.

Group 3- Comparator agent:  standard eye lid wipes will be administered to four times a day.

Clinical Investigations in the Study:

1. Best Corrected Visual acuity (BCVA) to document the visual acuity.

2. Schirmer’s test to evaluate the tear secreted.

3. Tear-film Break Up time (TBUT) to see the status of tear film stability

IDRA ocular surface analyzer will be used for evaluating the following parameters for each patient

1. Non-invasive tear film break-up time

2. Tear meniscus height

3. Lipid layer interferometry

4. Eye blink quality

5. Infrared meibography to assess meibum quality, and grade of meibomian gland dysfunction

 

Clinical
parameters

Follow up & investigation timepoints

|Day 0

Day 15

Day 30

Day 60

Day 90

|Visual acuity (BCVA)

x

x

x

x

x

|Schirmers test (mm)

x

x

x

x

x

|TBUT (sec)

x

x

    |I

D

R

A

  Tear Meniscus

Height

x

x

x

x

x

|Lipid Layer Interferometry

x

x

x

x

x

|Eye Blink Quality

x

x

x

x

x

|Infrared Meibography

x

x

x

x

x

X : will be done

Adverse events

Monitoring of adverse events and ocular discomfort will be done at each follow up and appropriate measure will be taken by the clinician.

a.     Design of statistical analysis

a.

The findings will be reported as Mean ± Standard error of Mean (SEM) and represented as bar graphs. Differences in the various parameters between the groups will be tested for statistical significance by Mann–Whitney test using GraphPad Prism 8.0 (GraphPad Software, Inc., La Jolla, CA, USA). Correlation among the various study parameters will be determined by Spearman Rank correlation tests using MedCalc® Version 12.5 (MedCalc Software, Ostend, Belgium). P < 0.05 will be considered to be statistically significant.

 

Clinical
parameters

Follow up & investigation timepoints

Statistical tests to be done

|Day 0

Day 15

Day 30

Day 60

Day 90

|Visual acuity (BCVA)

x

x

x

x

x

  Chi2 test/

Fisher’s exact test

|Schirmers test (mm)

x

x

x

x

x

|TBUT (sec)

x

x

    |I

D

R

A

  Tear Meniscus

Height

x

x

x

x

x

|Lipid Layer Interferometry

x

x

x

x

x

|Eye Blink Quality

x

x

x

x

x

|Infrared Meibography

x

x

x

x

x

|IVCM

x

x

x

x

x

   Secondary objective:

To understand the effect of 0.01% HOCL drops in (Neutrophil extracellular traps) NETs and ocular surface immune cell phenotype in dry eye and MGD.

**a.**Study design

 This will be a prospective molecular randomized trial.

 b.     Study area (multiple choice): Community/~~Hospital/~~Laboratory

 a.     Department of Ocular pathology: - All samples taken will stored and molecular experiments will be conducted here. Samples will be stored at -80°C freezer until use. Elisa, Flow Cytometry will be performed in the department’s laboratory.

 c.     Sample size estimation and sampling strategy

 At least 50 cases of Dry eye in each treatment group will be included for this objective.

 Tear Sample collection

Briefly, sterile Schirmer’s strips was used to collect the tear fluid. The collected strips were then stored in sterile microcentrifuge tubes at − 80 °C until further processing. Tear fluid proteins were eluted from the Schirmer’s strip by the agitation of cut pieces of Schirmer’s strip in 300 μl of sterile 1× PBS for 2 h at 4 °C followed by centrifugation. The eluted tear fluid (300 μl) was collected in a fresh sterile microcentrifuge tube was used for further downstream analyses.

 Ocular Surface wash collection

Briefly, open eye ocular surface wash samples were collected by an ophthalmologist in an outpatient clinical setup. A needleless sterile syringe was used to gently rinse the study subject’s ocular surface with sterile saline (room temperature). We used a sterile tube positioned close to the lateral canthus of the eye being irrigated to collect the runoff saline. Subsequently, 0.05% paraformaldehyde was used to fix the ocular surface wash samples and stored at 4 °C until further processing.

Impression cytology

 Ocular surface cells were collected non-invasively by impression cytology using an impression cytology paper at the 1st, 30th and 90th visit for each subject as per the manufacturer’s instructions. The sampling area of impression cytology was central bulbar conjunctiva inferior and proximal to the cornea. After the impression cytology sampling, the membrane from the device with the cellular material was ejected and collected in a 1.5-mL microcentrifuge tube. Two-thirds of the membrane of the impression cytology device was cut and immediately immersed in pre-chilled phosphate-buffered saline (PBS, pH 7.4) with fixative (0.05% paraformaldehyde) in 1.5-mL microcentrifuge tubes and stored at 4°C until further processing.

 d.     Project implementation plan

 

Investigation

Ttimepoints

|Day 0

Day 15

Day 90

|Sampling for ELISA

x

x

x

|Sampling for Flow

x

x

x

|

 Flow cytometry to determine ocular surface immune cell phenotype on ocular wash:

The proportions of various immune cell subsets on the ocular surface of study subjects will be determined by flow cytometry-based immunophenotyping using immune cell type-specific fluorochrome-conjugated antibodies. Briefly, the stored ocular surface wash samples/ impression cytology cells will be centrifuged at 2000 rpm for 5 min at 4 °C. The cell pellet from the ocular surface wash sample will be incubated with immune cell type-specific fluorochrome-conjugated antibody cocktails diluted in staining buffer (5% Fetal Bovine Serum in 1× Phosphate Buffer Saline, pH 7.4) with agitation (500 rpm) for 45 min at room temperature. Post incubation, the cells will be washed and resuspended in 300 μl 1× Phosphate Buffer Saline, pH 7.4.

Fluorochrome-conjugated antibodies specific for the various immune cell subtypes will be used. fluorochrome-conjugated antibodies will be procured from (BD Biosciences, USA). Data acquisition will be performed on a flow cytometer (BD FACSCantoâ„¢ II cell analyzer, BD Biosciences, USA) with BD FACSDiva software (BD Biosciences, USA) and acquired data will be analysed using FCS Express 6 (De Novo Software, USA). Post-acquisition compensation will be conducted using single stained controls. Further, specific cell populations will be identified, and regions will be assigned based on universal negative and fluorescence minus one control.

ELISA to detect the presence of Neutrophil extracellular traps (NETs) on tear samples

ELISA will be performed on the tear samples collected and stored in -20oC from the ocular surface of sub-chronic and chronic SJS patients. Roche duo set cell death detection ELISA kit (cat number -11544675001) will be used and protocol will be followed as mentioned in the kit manual.

e.     Design of statistical analysis

As per data normalization, parametric or non parametric test will be applied for statistical analysis