This study was an open-label, uncontrolled, phase 3, multicenter clinical trial designed to assess the efficacy and safety of ROH-101 in Japanese patients with CMV corneal endotheliitis. It was conducted across 13 sites from August 2021 to December 2022 with a trial structure that included a 2-week run-in period (from week − 2 to week 0), a 12-week treatment period (from week 0 to week 12), and a 24-week post-treatment observational period (from week 12 to week 36), as depicted in Fig. 1.

Study design. This study consisted of a run-in period for 2 weeks (from week − 2 to week 0), a treatment period for 12 weeks (from week 0 to week 12), and a post-treatment observation period for 24 weeks (from week 12 to week 36). The patients received 0.1% fluorometholone eye drops alone in the run-in period and in combination with ROH-101 in the treatment period. ROH-101 was discontinued in the post-treatment observation period, but corticosteroid eye drops were continued as needed

The trial was strictly conducted according to the Declaration of Helsinki principles, Good Clinical Practice guidelines, and the Act on Securing Quality, Efficacy, and Safety of Products Including Pharmaceuticals and Medical Devices. Before the study, and as necessary during its course, the ethics committees reviewed and approved the study protocol and informed consent form. Participation in the study was contingent upon each patient providing written informed consent to ensure that ethical standards were maintained throughout the trial. The study was registered with the Japan Registry of Clinical Trials under the identifier jRCT2051210064.

The patients in this study were diagnosed with CMV corneal endotheliitis on the basis of the clinical manifestations and through PCR testing for CMV of the aqueous humor according to the diagnostic criteria for CMV endotheliitis [15]. The included patients also had CMV DNA copy numbers of at least 103 copies/mL, had an endothelial cell density (ECD) of 500 cells/mm2 or more, were aged 20 years or older at the time of consent, were capable of having aqueous humor sampled by AC puncture, and had provided written informed consent to participate in the clinical trial. Patients who had received anti-CMV agents within 4 weeks before the start of the observation were excluded from this study. The inclusion and exclusion criteria are outlined in Table 1.

The patients received 0.1% fluorometholone eye drops alone in the 2-week run-in period and were then started on ROH-101 (a 0.15% ganciclovir gel) for the 12-week treatment period. During that time, 0.1% fluorometholone was continued in combination with ROH-101. The ROH-101 was administered as a single drop 5 times daily (once every 2–3 hours as the standard use), and the 0.1% fluorometholone eye drops were administered at 1–2 drops 4 times daily (once every 3 hours as the standard use). The ROH-101 was discontinued in the post-treatment observation period, whereas the corticosteroid eye drops were continued as needed.

During the study period, the concomitant use of topical and systemic antiviral agents, such as GCV, valganciclovir, letermovir, foscarnet sodium hydrate, acyclovir, valacyclovir, famciclovir, vidarabine, and amenamevir, was prohibited to avoid interference with the evaluation of the efficacy and safety of ROH-101. Similarly, the use of immunosuppressants and corticosteroids, with the exception of the 0.1% fluorometholone eye drops, was restricted up to week 12 or until treatment discontinuation. These concomitant prohibitions were established to ensure an accurate evaluation of ROH-101 without any confounding effects from other systemic or local therapies.

The primary endpoint of this study was the proportion of patients achieving a CMV DNA copy number in the aqueous humor of less than 103 copies/mL at week 12. Secondary endpoints included changes in CMV DNA copy numbers in the anterior humor, clinical findings observed by slit-lamp examinations (corneal edema, coin-shaped lesions, linear keratic precipitates [KPs] similar to rejection lines, other forms of KPs, and AC inflammation), and assessments of the best-corrected visual acuity (BCVA), IOP, ECD, and central corneal thickness (CCT) throughout the study period.

The participants were scheduled for a total of 11 visits, ranging from week − 2 to week 36. Comprehensive ophthalmologic evaluations, including slit-lamp examination, BCVA, IOP, ECD, and CCT measurements, were conducted at each visit and upon discontinuation of the study. PCR testing for CMV DNA in the aqueous humor was performed at critical time points: at baseline (week − 2), then at weeks 0, 4, 12, 36, and upon study discontinuation, as illustrated in Fig. 1.

Safety assessments included monitoring for adverse events (AEs) and evaluation of vital signs (heart rate and blood pressure) and laboratory test results. AEs were documented at every visit throughout the treatment and post-treatment observation periods. The AEs were categorized according to the primary system organ classes and preferred terms, using the Japanese version of the Medical Dictionary for Regulatory Activities (MedDRA, v 25.1).

Approximately 100 µL of aqueous humor was collected from the AC of each eye via AC puncture. The CMV DNA copy numbers were quantified by LSI Medience Corporation (Tokyo, Japan) by use of real-time PCR. The methodologies employed were consistent with those developed by Miyazaki and colleagues at Tottori University [22,23,24]. The process commenced with DNA extraction and was followed by amplification targeting the glycoprotein B gene of CMV by use of the LightCycler 480 II system (Roche). Fifty microliters of aqueous humor was extracted for DNA by use of the QIAamp DNA Mini Kit (Qiagen). The extracted DNA was eluted with 50 µL of water. Then, 8 µL of the extracted DNA was added to each PCR tube. The copy number was determined on the basis of the established dilutions of cloned glycoprotein B DNA. A threshold of less than 1.0 copies/µL (103 copies/mL) was set to designate a sample as CMV DNA negative on the basis of the results of validation testing by LSI Medience Corporation. The standard curve for the validation testing is shown in Table S1.

Statistical analyses for this study were conducted using SAS software, version 9.4 (SAS Institute). The required sample size was estimated by setting of parameters with a control rate of 23%, an expected success rate of 67%, a first-type error of 0.05 (2-sided), and a second-type error of 0.20 while using a binomial test for the null hypothesis. This calculation resulted in a target sample size of 12 patients.

The study included patients with CMV corneal endotheliitis who were treated with ROH-101 and assessed for efficacy at least once in the full analysis set (FAS), per-protocol set (PPS), and safety analysis set (SAF).

The primary endpoint was calculated as the rate of patients showing a decrease in CMV DNA copy numbers in the anterior humor to less than 103 copies/mL at the end of treatment (week 12). A binomial test was then performed with a threshold of 23% and a type 1 error (2-tailed) of 0.05 as the null hypothesis. We calculated the 95% CIs using Sterne’s methods.

The secondary endpoints were calculated as the frequency distribution (number of patients, percentage) of improvements in corneal edema, coin-shaped lesions, linear KPs, other forms of KPs, and AC inflammation at each assessment time point from baseline (week 0). The summary statistics (means and standard deviations [SDs]) were also calculated for the CMV DNA copy number changes in the aqueous humor and for IOP, ECD, CCT, and BCVA. The samples below the cut-off (103 copies/mL) for the CMV DNA copy number were treated as 0 copies/mL (logarithmically treated as 1).