This was a randomized, triple-blind, phase II clinical trial. The protocol was approved by the Institutional Review Board (CEI-2019/04/01). This trial was registered obtaining ClinicalTrial.gov Identifier: NCT04115800. Patients between 18 and 70 years old, with moderate-to-severe DED based on an Ocular Surface Disease Index (OSDI) score >22 and a van Bijsterveld score >4 at least in one eye, were invited to participate. Patients with past medical history of refractive surgery, ophthalmic surgery in the last 6 months, facial paralysis, lagophthalmos, herpetic keratitis, recent topical corticosteroids, history of oral isotretinoin in the previous 6 months, pregnant or lactating women, or known allergy or intolerance to sirolimus were excluded. During the study, patients were instructed to continue their current DED treatment, excluding topical corticosteroids, as well as to avoid introducing new medications throughout the trial.
All the medical staff at Institute of Ophthalmology "Conde de Valenciana" were informed of patient eligibility criteria. Candidates had to be referred to Department of Cornea and Refractive Surgery in order to begin enrollment. After complete explanation of the protocol and once they signed the informed consent form, the subjects were enrolled to the assay.
Ophthalmologic examination and baseline diagnostic studies were performed, including corrected distance visual acuity (CDVA) (LogMAR), OSDI score, conjunctival hyperemia grading (0-++++), tear osmolarity (mOsm/L) (TearLab Corporation, San Diego, California, USA), Schirmer s test with anesthesia (mm), corneal/conjunctival staining score (van Bijsterveld), intraocular pressure (mmHg), and LacryDiag testing (non-invasive tear break-up time (NITBUT) (seconds), lipid layer interferometry (nm), tear meniscus height (mm), meibomian gland dropout (%) (LacryDiag, Quantel Medical, Cournon-d'Auvergne, France) and detection of Matrix metalloproteinase-9 (MMP-9) protein levels of human tears (InflammaDry, Quidel Corporation, San Diego, CA, USA).
After concluding initial diagnostic tests, patients were randomized into two different groups using block randomization: Sirolimus liposomal formulation/liposome-loaded sirolimus (Blue) and liposomes without sirolimus/Sham (Green). This color code was used because the vials of liposomal suspension with and without sirolimus were prepared and labeled by only one of the researchers in the laboratory with Good Manufacturing Practice (GMP) resources, using blue labeled vials for those containing sirolimus liposomal formulation and green labels for those containing liposomal suspension without sirolimus. This investigator was not involved in the clinical assay and the analysis until the trial data were opened. All other investigators and participating patients were blinded to the components and preparations.
Both sirolimus-loaded suspension and liposomal suspension without sirolimus were prepared with GMP by Laboratorio Santgar Formulas Magistrales de Mexico SA de CV, using the Garcia-Sanchez Gustavo A. and Bernad Bernad Maria J. patented technique described in "IMMUNOSUPPRESSSIVE TREATMENTS FORMULATION AND METHODS" United States Patent Number: 9801862 B2 and World Intellectual Property Organization, International Publication Number: WO 2015/068020 A2. The vials were kept at 2-8 C until dispensed to the patients. Briefly, primary formulation comprises a suspension of approximately 0.4 mg/mL sirolimus encapsulated in soybean lecithin, cholesterol and sucrose liposomes, the aqueous phase comprised in a balanced (pH 7.4) buffer. Treatment was applied as the last step of the first visit, after initial diagnostic studies and patient classification. Subconjunctival injections were applied under topical anesthesia (tetracaine solution 5mg/mL, Ponti Ofteno, Laboratorios Sophia, Mexico). Under the slit-lamp, a 27-gauge syringe was used for injection 60 microg/0.15mL of the indicated suspension in the superior temporal subconjunctival space 4mm away from the corneal limbus. Both eyes of each patient were injected in every visit.
Two follow-up visits (second and third visits) were scheduled, each with a 10+-2 days interval. In those visits, only the OSDI questionnaires were repeated and patients were inquired about possible adverse effects. At the end of the visit, the second and third doses were applied respectively, using the aforementioned technique.
At the fourth and last follow-up visit (approximately 4 weeks after baseline/first visit), patients underwent ophthalmologic examination and ancillary studies performed at the baseline examination for comparisons.
Information was collected and recorded in a Microsoft EXCEL 2013 database, where missing, extreme or inconsistent data were identified and adjusted through database verification. Once all the information was verified, descriptive statistics analysis was performed, reporting frequencies and proportions for qualitative variables, along with means, standard deviations, medians, interquartile ranges, statistical dispersions and central tendency measures for continuous variables.
Statistical analysis was performed using Prism 8.3.1 Software. Kolmogorov-Smirnov and Anderson-Darling tests were initially performed to determine data normal distribution. Differences between groups regarding pre- and post-treatment variables were evaluated through X tests and non-paired t-tests, whereas intra-group (pre- and post-treatment) differences were assessed using paired t-tests. A p-value less than 0.05 was considered statistically significant.
This trial was reviewed and approved by the Institute of Ophthalmology "Conde de Valenciana" Ethics Committee (CEI-2019/04/01) and was performed following the ethical procedures established by the Declaration of Helsinki. Informed consent included all the data necessary for patients to make an informed decision, and all participating patients signed the informed consent form before inclusion in the study.