This was a phase 3, double-masked, intra-patient randomised, placebo-controlled trial of 0.5% epinastine topical eyelid cream in patients with a history of seasonal allergic conjunctivitis. The study was conducted at one clinical site (Kitasato University Kitasato Institute Hospital, Tokyo, Japan) between May 13 and July 12, 2022. The study was conducted in compliance with ethical principles based on the Declaration of Helsinki as revised in 2013, standards set forth in the Japanese Pharmaceuticals and Medical Devices Act and Ministerial Ordinance on Good Clinical Practice. The Institutional Review Board reviewed and approved the study protocol before trial commencement, and all patients provided written informed consent to participate. This study is registered with the Japan Registry of Clinical Trials (JRCT ID: jRCT2031220074, available at https://jrct.niph.go.jp/latest-detail/jRCT2031220074).

Eligible patients were asymptomatic adults (aged 20–65 years) with a history of seasonal allergic conjunctivitis. Key exclusion criteria included comorbid inflammatory eye conditions or dry eye disease on the ocular surface or anterior segment, recent intraocular surgery (≤ 90 days before screening) or recent treatment for lacrimal punctum occlusion (≤ 30 days before screening). Patients treated with oral corticosteroids ≤ 28 days before screening, or treated with anti-allergic drugs, H1-receptor antagonists or non-steroidal anti-inflammatory drugs ≤ 7 days before screening were also excluded. Additional eligibility criteria for this study are provided in Table 1.

The study comprised a screening period of ≥ 22 days followed by a treatment period of 2 days (Fig. 1). Eligible patients entered the screening period ≤ 30 days after providing written informed consent; if the screening period began > 30 days after the date of consent, written informed consent was re-collected.

Design of the clinical trial, showing patient visits and treatment periods. CAC, conjunctival allergen challenge; IgE, immunoglobulin E

At the start of the screening period (visit 1), eligible patients were tested for Japanese cedar pollen allergy using a serum antigen-specific immunoglobulin E (IgE) antibody test; those who tested negative were excluded from the study. At visit 2 (21 days before the start of the treatment period), conjunctival allergen challenge (CAC) tests were performed to determine the optimal concentration of allergen solution required to trigger allergic conjunctivitis symptoms.

Before the CAC tests, allergen solutions containing Japanese cedar pollen extract (25-fold, 50-fold, 100-fold and 200-fold dilutions) and negative control solutions containing glycerine (25-fold dilution) were prepared (Torii Pharmaceutical Co., Ltd.). All diluted allergen solutions contained glycerine. At visit 2, one drop of negative control solution (without allergen) was instilled in each eye, and patients with a conjunctival inflammatory response were excluded from the study.

At visit 3 (14 days before the start of the treatment period), a confirmatory CAC test was performed using the optimal allergen concentration determined at visit 2. After antigen challenge in both eyes, the severity of ocular itching was assessed at 3, 5 and 10 min, and the severity of bulbar conjunctival hyperaemia was assessed at 5, 10 and 20 min. The optimal allergen concentration was confirmed if ocular itching and bulbar conjunctival hyperaemia severity scores were ≥ 2 in each eye. Throughout the screening period, patients were required to show no signs or symptoms of ocular itching or conjunctival hyperaemia before the CAC tests, and those unable to achieve ocular itching and bulbar conjunctival hyperaemia severity scores ≥ 2 in both eyes after antigen challenge were excluded from the study.

On day 1 of the treatment period (visit 4), the left and right eyes of each patient were randomised to receive either 0.5% epinastine topical eyelid cream (approximately 30 mg) [23] or placebo (base cream without epinastine hydrochloride). Eyes were randomly assigned to study cream or placebo using a permuted block method with a block size of 4. Because this was a double-masked study, the study cream and placebo were packaged in identical tubes, and patients and investigators were masked to the treatment allocations for each eye.

At visit 4, study investigators instructed patients to draw a single dose (approximately 1.3 cm of cream as a guide) of 0.5% epinastine topical eyelid cream/placebo cream onto the fingertip to apply onto the periocular area (outer skin of the upper and lower eyelids) of the assigned eye. On day 2 of the treatment period (visit 5; 24 h after application of the study cream and placebo), a CAC test was performed in each eye using the optimal allergen concentration that was determined during the screening period. The severity of ocular itching and bulbar and palpebral conjunctival hyperaemia were assessed after antigen challenge as performed at Visit 3 (Table 2).

The primary efficacy endpoints were a 3-time point mean ocular itching score (the average of scores obtained at 3, 5 and 10 min after the antigen challenge) and a 3-time point mean conjunctival hyperaemia score (sum of the bulbar and palpebral conjunctival hyperaemia scores; averaged over 5, 10 and 20 min after the antigen challenge) in each eye on day 2 of the treatment period (24 h after treatment application, in line with the expected once daily administration). Secondary efficacy endpoints included the mean ocular itching, conjunctival hyperaemia, palpebral conjunctival hyperaemia and bulbar conjunctival hyperaemia scores over time during day 2. Treatment effects were assessed by performing CAC tests 24 h after the single-dose application of the study cream and placebo. Safety endpoints included the incidence and severity of adverse events (AEs) and adverse drug reactions (ADRs) during the treatment period, and intraocular pressure (IOP) and funduscopy assessments throughout the screening and treatment periods.

A sample size of 30 patients (60 eyes) was estimated to provide 90% power to detect the superiority of 0.5% epinastine topical eyelid cream versus placebo for the primary endpoints. This was calculated assuming mean ± standard deviation (SD) differences in ocular itching and conjunctival hyperaemia scores of 1.0 ± 0.5 and 1.0 ± 1.6, respectively, paired t-tests, type I error rate of 5% and power of 90%.

Baseline demographics and clinical characteristics (measured at visit 1) were based on the intention to treat (ITT) population and summarised using descriptive statistics (means, SDs, medians, ranges, number of patients and/or proportions as appropriate). Efficacy analyses were based on the full analysis set (FAS), defined as all randomised patients who had received ≥ 1 application of the study cream or placebo and for whom efficacy data were available.

Primary efficacy endpoints were summarised using least squares (LS) means, standard errors (SE), mean differences and 95% confidence intervals (CIs). Secondary efficacy endpoints were summarised using means and SDs. The analysis of the primary endpoints was performed on data from the FAS using a linear mixed-effect model, with treatment as the fixed effect and patient as the random effect.

The safety analysis population included all patients who had received ≥ 1 application of study cream or placebo and had available safety data. Safety was assessed through descriptive summaries of AEs, ADRs, IOP and fundoscopy findings by treatment group. AEs and ADRs were coded using the Medical Dictionary for Regulatory Activities’ thesaurus terms and summarised by Preferred Term and System Organ Class. Statistical analyses were performed using SAS version 9.4 or later, and efficacy analyses used a two-tailed significance level of α = 0.05.