Background and Objectives <p>Based on melatonin’s neuroprotective effects in pre-clinical multiple sclerosis models, the MELATOMS-1 study was designed to evaluate melatonin treatment in patients with primary progressive multiple sclerosis (PP-MS) receiving ocrelizumab treatment. The trial was prematurely halted due to hypertransaminasemia. This study aimed to analyse observed cases of hypertransaminasemia and explore potential underlying mechanisms, focusing on drug–drug interactions.</p> Methods <p>This study reports findings from MELATOMS-1 (NCT03540485), a multicentre, phase I/II, randomised, double-blind, placebo-controlled trial conducted in the multiple sclerosis units of Hospital Universitario Virgen Macarena, Hospital Universitario Virgen del Rocío and Hospital Vithas Nisa of Seville. The trial was designed to evaluate the safety and efficacy of high-dose oral melatonin (300 mg/day) as an adjunct therapy for patients with PP-MS (Expanded Disability Status Scale 2–7) on stable ocrelizumab therapy (&gt; 9 months). Participants were assigned 1:1 by stratified randomisation (based on MS severity score) to receive either daily oral melatonin or a matching placebo 30 min before bedtime. Safety was evaluated by monitoring adverse events and scheduled biochemical analyses, including routine liver function tests [alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and bilirubin, quantified by automated immunoassay], every 3 months for up to 2 years (the trial’s endpoint). The trial was temporarily stopped after grade 1–2 hepatotoxicity was identified in three patients, according to the scale of the international DILI expert working group. A subsequent post hoc causality analysis focused on potential drug–drug pharmacokinetic interactions between high-dose melatonin and the patients’ polypharmacy involving cytochrome P450 (CYP) enzyme pathways. The analysis focused on concomitant medications including acetaminophen, metamizole, omeprazole, ibuprofen, acetylsalicylic acid, nabiximol and tizanidine.</p> Results <p>The trial was prematurely stopped and unblinded after eight patients had been recruited. Three out of the four patients receiving melatonin developed hypertransaminasemia, which resolved after treatment discontinuation. All affected patients were women taking polymedications metabolized through shared hepatic pathways with melatonin, suggesting a possible interaction leading to hepatic overload. In contrast, the only male participant in the arm, who did not take medications that shared metabolism with melatonin, experienced no adverse liver-related events during his 14-month treatment period.</p> Conclusions <p>Despite the fact that melatonin has a good safety profile, these findings raise concerns regarding the hepatotoxic potential of high doses of melatonin in polymedicated patients. This is attributed to a probable pharmacokinetic drug–drug interaction with concomitant medications sharing liver metabolization pathways with melatonin, leading to CYP450 metabolic pathways saturation. Though these findings should be interpreted with caution due to the small sample size and heterogeneity of the study population, and further studies are needed to elucidate the underlying mechanisms and establish safety guidelines, this study reveals a critical safety event that requires careful consideration when designing future clinical trials involving high-dose melatonin, especially in polymedicated populations.</p> Clinical Trial Number <p>NCT03540485.</p>