Hepatic Safety of Adjunctive High-Dose Melatonin in Participants Receiving Ocrelizumab for Primary Progressive Multiple Sclerosis: Liver Toxicity Findings from a Phase I/II Randomised Clinical Trial (MELATOMS-1)

Abstract

Background and Objectives

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 .

Methods

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 (> 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.

Results

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.

Conclusions

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.

Clinical Trial Number

NCT03540485.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40263-025-01261-w.

Plain Language Summary

Melatonin is a hormone that helps regulate sleep and has been studied for its potential benefits in multiple sclerosis (MS). The MELATOMS-1 study was designed to test the safety and efficacy of melatonin treatment in patients with primary progressive MS who are already being treated with ocrelizumab, a drug that changes the immune system. The patients were randomly assigned to take 300 mg of melatonin every day or placebo (a dummy pill without active ingredients). The study was prematurely discontinued after 14 months and eight patients had been enrolled, due to the development of liver toxicity in three of the four patients randomised to receive melatonin. These adverse events disappeared after stopping melatonin. All affected patients were women taking multiple medications, many of which are processed by the liver in the same metabolic pathway as melatonin. In addition to the four placebo-treated patients, the only melatonin-treated patient who did not experience any related toxicity after 14 months of treatment was a male who did not take other drugs with the same metabolic pathway as melatonin. These findings suggest that high doses of melatonin may cause liver toxicity if combined with other medications that share the same processing pathway in the liver. Owing to this, the study was modified to continue testing melatonin at a lower dose to find dose-limiting toxicity. More research is needed to determine how melatonin interacts with other drugs and how it can be safely used in patients with MS.

Supplementary Information

The online version contains supplementary material available at 10.1007/s40263-025-01261-w.

Key Points

In the MELATOMS-1 clinical trial, all hypertransaminasemia cases occurred in women receiving drugs metabolized via hepatic pathways shared with melatonin, suggesting a potential sex-related predisposition to melatonin-induced drug-drug interaction.

The only patient without hypertransaminasemia was a man who did not take medications metabolised through the same liver pathways as melatonin, suggesting that drug interactions contributed to hepatotoxicity. This finding underscores the need for further research to identify subgroups of patients at increased risk for adverse events.

The resolution of liver enzymes alteration after discontinuing melatonin, along with the fact that all affected patients were taking multiple medications metabolized by melatonin-shared liver pathways, highlights the need to monitor liver function and drug interactions in polymedicated patients receiving high doses of melatonin.

Introduction

Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disorder of the central nervous system (CNS), characterised by demyelination, axonal damage and progressive neurological disability [2]. Primary progressive multiple sclerosis (PP-MS) is a particularly challenging subtype of MS owing to its steady clinical decline and limited treatment options [3]. PP-MS accounts for approximately 10–15% of all MS cases and is distinguished by a gradual worsening of neurological function from the onset, without distinct relapses or remissions [4]. Current therapies, such as ocrelizumab, a monoclonal antibody targeting CD20+ B cells, have shown efficacy in slowing disease progression in PP-MS [4, 5]. However, it is unknown whether adjunct therapies can further modulate neuroinflammation and neurodegeneration.

Melatonin is a pleiotropic molecule that, beyond its well-known function as chronoregulator, has garnered attention for its anti-inflammatory and anti-oxidant properties [6–9], making it a promising candidate for the treatment of neurodegenerative and autoimmune diseases. Preclinical studies using the experimental model of autoimmune encephalomyelitis (EAE), a primary preclinical model of MS, have demonstrated that melatonin reduces inflammatory responses and enhances the suppressive capacity of regulatory CD4+ T cells in the CNS, leading to improved clinical outcomes [10–12]. In patients with relapsing-remitting MS (RR-MS), melatonin has been shown to decrease inflammatory responses in peripheral T lymphocytes, suggesting a potential role in immune modulation [13]. In addition, its antioxidant effects [14] may help to mitigate the neuronal damage in MS. Oxidative stress drives mitochondrial dysfunction, DNA damage and lipid peroxidation, all of which contribute to axonal degeneration and disease progression [3]. By scavenging free radicals and enhancing the activity of endogenous antioxidant enzymes, melatonin can protect neurons and oligodendrocytes from oxidative damage [15]. These properties point to melatonin as a potential therapeutic agent to slow the neurodegenerative processes that drive the progression of disability in PP-MS [16].

In general, reports of adverse events in studies involving high-dose melatonin have been sparse and the available evidence suggests that melatonin has a favourable safety profile [17–20]. However, as the use of melatonin expands beyond its conventional role as a sleep aid, further research is necessary to fully understand its physiological effects and the range of potential adverse outcomes. Therefore, long-term trials are needed to improve the quality and consistency of safety reporting.

Based on melatonin properties, we hypothesized that the administration of this molecule to patients with PP-MS receiving ocrelizumab (the only treatment specifically approved for PP-MS by the EMA and authorised in Spain) would potentiate neuroprotective and anti-inflammatory benefits. Specifically, we anticipated that melatonin would modulate neuroinflammation markers, reduce oxidative stress and slow disability progression in these patients.

The primary objective of the MELATOMS-1 study was to evaluate the safety and efficacy of high-dose melatonin (300 mg/day) in patients with PP-MS treated with ocrelizumab. The trial was stopped prematurely due to elevated liver transaminases. After protocol amendment, the trial was reinitiated and is ongoing. It encompasses a reduced initial dose of 100 mg melatonin and a structured dose de-escalation regimen (50, 25, 10 mg) in case of hypertransaminasemia, as well as increased safety surveillance. The present publication reports the findings regarding liver toxicity that caused the trial to be stopped.

Materials and Methods

Study Design

The participants described here were enrolled in the MELATOMS-1 study (ClinicalTrials.gov Identifier: NCT03540485; EUCT number: 2023-508457-10-00, previously 2018-001779-18 [EudraCT Number]), a multicentre phase I/II randomized, double-blind (investigators, participants and caregivers), placebo-controlled clinical trial to assess the safety and efficacy of melatonin. Participants were assigned in a 1:1 ratio to the melatonin group or the placebo group. To ensure that each comparison group had an equal number of participants with similar baseline characteristics in terms of Expanded Disability Status Scale (EDSS) and time since disease diagnosis, a stratified randomisation was performed on the basis of the severity of the disease. In particular, stratification was carried out using the Multiple Sclerosis Severity Score (MSSS), dividing the participants into two blocks: block 1 (MSSS between 0 and 5) and block 2 (MSSS between 5 and 10).

The motivation to use 300 mg daily melatonin as an intervention in this study was based on robust preclinical and clinical evidence supporting both the efficacy and safety of high-dose melatonin regimens and their ability to achieve pharmacologically relevant concentrations in the central nervous system (CNS). Preclinical studies [10, 12] demonstrated that melatonin at 80 mg/kg/day, which is a dose equivalent to 450 mg/day in humans (assuming an average body weight of 70 kg), according to [21], exerts potent immunomodulatory and neuroprotective actions in the EAE model, where it attenuates neuroinflammation, enhances regulatory T cell function and mitigates oxidative stress and demyelination, leading to improved clinical outcomes. Clinically, López-González et al. [22] reported a case of a patient with primary progressive multiple sclerosis (PP-MS) treated with 300 mg/day of oral melatonin for 4 years, who exhibited a notable clinical improvement in disability scores without any reported adverse events, further supporting the safety and therapeutic potential of this high-dose regimen in humans. In addition, other studies using comparable or even higher doses of melatonin (up to 1–3 g/day) in different populations have confirmed its favourable tolerability profile, the absence of hepatotoxicity and stability of the biochemical parameters during long-term administration (summarised in Supplementary Table S1).

Participants

The key eligibility criteria were an age between 18 and 65 years, a diagnosis of PP-MS (according to McDonald’s diagnostic criteria modified in 2010), an EDSS score of 2 to 7 at the time of screening and no immunomodulatory or immunosuppressive treatment during the 3 months before participation in the trial (except ocrelizumab in stable doses for at least 9 months before inclusion in this study). The key exclusion criteria were an alternative diagnosis that explains both neurological disability and findings in nuclear magnetic resonance; a clinical history of hypersensitivity reactions to melatonin; pregnancy or lactation; and abnormal results in basal blood tests. This study included those participants recruited until the clinical trial was interrupted.

Safety Monitoring and Procedures

During the course of the trial, participants’ safety was monitored through scheduled clinical evaluations, laboratory tests and systematic follow-up of all adverse events. Safety evaluations included physical examination, vital signs measurement and complete laboratory testing of blood and urine samples. Blood evaluations comprised a full hematologic panel (complete blood count), general biochemistry profile (including electrolytes, renal function markers, glucose and C-reactive protein [CRP]), liver profile (alanine aminotransferase [ALT], aspartate aminotransferase [AST], gamma-glutamyl transferase [GGT], alkaline phosphatase [ALP], total and direct bilirubin, and CRP) and lipid profile (total cholesterol, HDL, LDL and triglycerides). Urinalysis included standard parameters for renal and metabolic function. These evaluations were performed at baseline and at predefined intervals throughout the study period (3, 6, 12, 18 and 24 months), as well as whenever clinically indicated to ensure continuous monitoring of participant safety.

In addition to scheduled study visits, adverse events were actively monitored during interim telephone contacts with participants. Any adverse event spontaneously reported by a participant or identified through review of available medical records, whether occurring within or outside of predefined study visits, was documented and evaluated. This approach ensured continuous surveillance of potential safety concerns throughout the entire follow-up period.

Any subject presenting with an adverse event was followed until full resolution or stabilisation. If laboratory abnormalities were clinically significant or associated with symptoms that met the definition of a serious adverse event, the diagnosis was recorded and reported as an SAE (may cause death, threaten the life of the subject, require hospitalisation or prolong an existing hospitalisation, cause permanent or significant disability or incapacity, or result in a congenital anomaly or malformation. Suspected medically significant adverse events or adverse reactions, even if they do not meet the above criteria, were also considered serious. These include significant medical events that require intervention to prevent one of the consequences described above. Likewise, all suspected transmission of an infectious agent through a medicinal product would be reported as serious). The sponsor’s pharmacovigilance unit ensured the collection and reporting of all SAE during the study period in accordance with current legislation. Annual safety reports and suspected unexpected serious adverse reactions (SUSARs) were submitted to the Spanish Agency for Medicines and Medical Devices (AEMPS), regional health authorities and the Research Ethics Committee within the required timelines.

The evaluation of the intensity of liver toxicity was carried out according to the Common Terminology Criteria for Adverse Events (CTCAE, version 5.0). Reference thresholds and classification criteria for liver enzyme elevations (ALT, AST, GGT, ALP and bilirubin) were applied to ensure consistent classification of adverse liver events. Simultaneously, information on liver enzyme alterations described in the current Summary of Product Characteristics (SmPC) of melatonin at the approved therapeutic dose was reviewed and considered to contextualise the observed findings and support the causality assessment.

Following the temporary discontinuation of the study due to cases of elevated liver transaminases, additional safety activities were implemented. The Pharmacovigilance Unit, in collaboration with the global project management team, conducted an extensive literature review addressing safety and dosing aspects of the investigational product, as well as a thorough re-evaluation of all available patient data. This review included an analysis of individual clinical histories, concomitant medications, exposure to treatment and all relevant laboratory and imaging results.

The findings were discussed in a panel of multidisciplinary clinical experts (neurologists, hepatologists, biochemists and clinical pharmacologists) to obtain expert clinical input and guide future risk mitigation actions, including consideration of a possible dose reduction in subsequent study phases. In this way, after the temporary suspension of the trial due to elevated liver transaminases, the protocol was amended and approved by the regulatory authority and ethics committee. The trial was resumed at a reduced dose of 100 mg melatonin, with updated patient information and informed consent, intensified safety monitoring (including four additional visits at months 1, 9, 15 and 21), and a structured dose de-escalation scheme (50, 25, 10 mg). Predefined stopping rules for mild hepatotoxicity were according to international DILI criteria (ALT ≥ 5 × ULN, ALP ≥ 2 × ULN, or ALT ≥ 3 × ULN with total bilirubin > 2 × ULN). Participants with treatment interruptions underwent biweekly laboratory evaluations during the first month and monthly up to 3 months, including liver function tests, general hematology test and viral serology, with clinical review by hepatology specialists as needed. Treatment could be resumed at a lower dose if liver enzymes normalised; persistent hepatotoxicity resulted in permanent withdrawal. The baseline exclusion criteria for ALT or AST were refined to 1.5 × ULN (previously 3 × ULN). Under this condition, 16 participants have been enrolled and the trial is still ongoing. To date, ongoing safety evaluations have not identified any safety concerns that would preclude the continuation of the study. However, increased vigilance remains in place and safety alerts will be activated immediately if new cases of elevated transaminases meet the criteria of DILI with a suspected causal relationship with the investigational product.

Following amendment approval, the re-enrolment of participants previously exposed to the investigational product at 300 mg was carefully evaluated by the clinical team, the sponsor and the participants. Only individuals who had experienced no serious adverse events during initial participation, met all updated eligibility criteria, including the more stringent hepatic laboratory thresholds and provided informed consent acknowledging and accepting the new study conditions were re-enrolled. Based on this assessment, two participants previously enrolled at 300 mg were re-enrolled at the new dose level (100 mg), one of whom had received 300 mg of the investigational product for approximately 1 year prior to the temporary study suspension.

Biochemical Measurements

Biochemical parameters (ALT, AST, GGT, ALP, bilirubin and CRP) were evaluated in serum from fasting blood samples as previously described [23–25] using an automated immunoassay electrochemiluminescence system (COBAS c702 and e602 analysers; Roche Diagnostic, Basel, Switzerland). White blood cells were quantified in blood samples using a SYSMEX XE 5000 fluorescence flow cytometer (Sysmex Europe GmbH, Norderstedt, Germany).

Results

After 14 months from the official initiation of the study, a total of eight patients were recruited and treated, with a follow-up ranging 2 to 14 months, three patients (37.5%) developed elevated liver enzymes within the first 3 months of treatment (cases 1, 2 and 3). The flow of participants through the study is illustrated in Fig. 1 [1]. This observation led to the unblinding and discontinuation of the study. The three cases with hepatotoxicity had been randomised to receive melatonin and were women, whereas the fourth patient who received melatonin was male, and after 14 months of treatment did not record any liver toxicity events. None of the four participants in the placebo group (a man and three women) exhibited comparable liver abnormalities. A timeline detailing the recruitment and treatment duration for all patients is provided on Fig. 2. The ages of the participants ranged from 38 to 59 years, with a mean duration of the disease of 10.5 years and an average EDSS score of 6.25 (Table 1). All patients received ocrelizumab as part of their standard treatment for PP-MS, together with various concomitant medications (Table 2). None of the participants had a history of significant liver disease, alcohol abuse or use of herbal agents prior to the trial.

Fig. 1.

CONSORT flow diagram of the MELATOMS-1 trial. Data correspond to the high-dose (300 mg/day) phase before protocol amendment and dose reduction

Fig. 2.

Patient recruitment schedule. The timeline indicates the dates of enrollment and duration of treatment for each patient. The 14 months of enrolment at the centre (February 2020–April 2021) are represented, with patients in the melatonin arm shown in pink and patients in the placebo group shown in purple. The dashed lines represent the period in which melatonin was discontinued in case 2. The symbol ↓ represents permanent withdrawal due to hypertransaminasemia

Table 1.

Patient characteristics

Experimental Group	Patients	Age (years)/Sex	EDSS	Years of disease	Months with ocrelizumab	Months of treatment
Melatonin	Case 1	57/Female	5.5	17	19	1
	Case 2	52/Female	6.5	5	11	3-discontinuation-1 (months 6–7)
	Case 3	46/Female	6.5	15	9	4
	Mel 4	38/Male	7	5	18	15
Placebo	Placebo 1	50/Female	6.5	5	27	13
	Placebo 2	51/Male	6.5	16	35	5
	Placebo 3	48/Male	6	14	13	4
	Placebo 4	59/Male	5	6	23	2

Table 2.

Concomitant medication for each patient in the MELATOMS-1 trial

Patient case	Medication	Dose
Case 1	Acetaminophen	500 mg occasionally
	Dalfampridine	10 mg twice a day
	Metamizole	575 mg/day
Case 2	Baclofen	10 mg/day
	Dalfampridine	10 mg twice a day
	Ibuprofen	600 mg on demand
	Omeprazole	20 mg/day
	Sertraline	50 mg/day
Case 3	Acetaminophen	1 g every 8 h
	Acetylsalicylic acid	100 mg/day
	Cefadroxil	500 mg/day
	Dalfampridine	10 mg twice a day
	Denosumab	60 mg every 6 months
	Hidroferol	1000 UI/day
	Nabiximol	4 sprays/day
	Omeprazole	20 mg/day
	Oral calcium	600 mg/day
	Tizanidine	4 mg twice a day
Mel 4	Baclofen	10 mg/day
	Dalfampridine	10 mg twice a day
	Hidroferol	0.266 mg/day
	Mirabegron	25 mg/day
	Propiverine	45 mg/day
	Tamsulosin	0.4 mg/day
Placebo 1	Baclofen	10 mg three times a day
	Daflon	500 mg twice a day
	Fampridine	10 mg twice a day
	Fesoterodine	4 mg/day
	Gabapentin	300 mg/day
	Levothyroxine	112 µg/day
	Mirabegron	50 mg/day
	Sertraline	50 mg/day
	Tamsulosin	0.4 mg/day
Placebo 2	Calcifediol	266 µg/day
	Fampridine	10 mg twice a day
	Magnesium	48.62 mg/day
	Rosuvastatin	20 mg/day
Placebo 3	Amantadine	100 mg twice a day
	Baclofen	12.5 mg three times a day
	Calcifediol	266 µg/day
	Diazepam	10 mg/day
	Escitalopram	20 mg/day
	Enalapril	12.5 mg/day
	Fampridine	10 mg twice a day
	Tamsulosin	0.4 mg/day
	Vitamine B9/B12	400/2 µg/day
Placebo 4	Acetaminophen	1 g occasionally
	Lorazepam	2 mg/day
	Sertraline	100 mg/day

According to the criteria proposed by the international drug-induced liver injury (DILI) expert working group, case 1 experienced mild asymptomatic hypertransaminasemia with alanine aminotransferase (ALT) levels 7.4 times the upper limit of normal (ULN) and aspartate aminotransferase (AST) levels 3.6 times the ULN after 1 month of treatment. Liver enzymes normalised within 2 months after discontinuation of melatonin (Table 3).

Table 3.

Time course of biochemical variables during treatment in the melatonin group (reported on month basis)

		Case 1				Case 2						Case 3					Case 4
Melatonin group	Laboratory tests	Basal	1 ma	2 ma	3m	Basal	3 m	4 ma,b	6 m	7 ma	8 ma	Basal	3 m	4 ma	4.5 ma	5 ma	Basal	3 m	6 m	12 m
	ALT (10–40 U/L)	21.6	296	43	18	19	697	156	14	104	12	18	123	263	104	37	49	29	23	20
	AST (10–37 U/L)	20.1	144	26	19	17	725	287	18	118	17	24	54	141	55	37	26	19	17	12
	GGT (11–49 U/L)	21	30	33	19	35	581	425	72	218	94	13	30	41	40	nt	32	32	29	35
	ALP (40–130 U/L; men) (35–105 U/L; women)	134	nt	150	106	68	228	nt	85	127	104	34	40	46	43	nt	46	40	40	48
	Bilirubin (0.1–1.2mg/dL)	nt	nt	1.15	1.03	0.42	3.09	3.25	nt	0.83	nt	0.69	0.25	nt	0.38	0.47	0.68	0.87	0.84	nt
	CRP (0–5 mg/L)	1.1	10.25	0.96	0.77	3.5	12.2	nt	5.5	nt	12.6	0.6	3	nt	nt	nt	2.9	3.2	4.5	2.2
	MLT treatment	−	+	−	−	−	+	−	−	+	−	−	+	+	−	−	−	+	+	+

EDSS, Expanded Disability Status Scale; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamyltransferase; ALP, alkaline phosphatase; CRP, C-reactive protein; MLT, melatonin; nt, not tested; m, months

^aUnscheduled visit

^bMelatonin had been suspended just one day before

Case 2 had severe hypertransaminasemia (ALT: 17.4 × ULN; AST: 19.6 × ULN), accompanied by elevated bilirubin (3 × ULN) as well as clinical symptoms such as fatigue, jaundice and digestive discomfort. In addition, γ-glutamyltransferase (GGT) increased significantly (581 U/L), and alkaline phosphatase (ALP) was also elevated (228 U/L). These symptoms were observed in the 3-month follow-up biochemistry test. When this situation was detected, although the patient was in good general condition, melatonin was temporarily discontinued. At 1 day after suspension (4 months after starting treatment), blood tests were repeated and the transaminase levels decreased dramatically (Table 3). No complementary studies, such as serological tests for hepatitis A, hepatitis B, hepatitis C, Epstein–Barr virus (EBV), cytomegalovirus (CMV), autoimmune serology or imaging tests, were performed. Liver enzymes improved after discontinuation but increased again upon reintroduction of melatonin, leading to permanent withdrawal.

In case 3, an increase in ALT (3 × ULN) and AST (1.4 × ULN) was observed after 3 months of melatonin treatment. After 1 month, transaminase levels continued to rise (ALT: 6.5 × ULN; AST: 3.8 × ULN). Although the patient was asymptomatic, the treatment was eventually withdrawn, and the clinical trial was stopped and unblinded. After a month of melatonin discontinuation, ALT and AST levels returned to normal values (Table 3).

Therefore, the mobilization of liver enzymes in the three cases was temporally associated with the melatonin treatment. In case 2 specifically, the reintroduction of melatonin led to a recurrence of elevated liver enzymes, which further supports the causal relationship between melatonin and hepatotoxicity. No other potential causes of hypertransaminasemia, such as viral hepatitis or autoimmune liver disease, were identified in these patients (Supplementary Table S2).

In addition, C-reactive protein (CRP) levels increased in cases 1–3 during melatonin treatment (Table 3).

The fourth patient receiving melatonin (Mel 4) showed no substantial elevation of liver enzymes during the 14-month treatment period.

In the placebo group, no significant elevations in ALT or AST were observed in any patient, and all liver enzyme levels remained within normal ranges throughout the study (Table 4). GGT, ALP and bilirubin levels also remained stable in all patients, with no clinically significant changes reported during the trial. One patient showed a temporary increase in bilirubin (3.09 mg/dL), and another had CRP levels, but these were not associated with clinical symptoms.

Table 4.

Time course of biochemical variables during treatment in the placebo group (reported on month basis)

		Placebo 1				Placebo 2		Placebo 3			Placebo 4
Placebo group	Laboratory tests	Basal	3 m	6 m	12 m	Basal	3 m	Basal	1 m*	3 m	Basal
	ALT (10–40 U/L)	14	14	17	21	20	22	22	24	22	16
	AST (10–37 U/L)	17	20	20	24	25	26	16	nt	14	17
	GGT (11–49 U/L)	8	9	14	15	43	42	43	40	37	16
	ALP (40–130 U/L; men) (35–105 U/L; women)	nt	71	83	90	53	52	54	68	60	61
	Bilirubin (0.1–1.2 mg/dL)	0.22	0.28	0.27	nt	0.87	3.09	0.47	0.41	nt	nt
	CRP (0–5 mg/L mg/L)	0.3	0.3	0.5	1.9	0.8	nt	4.9	nt	7.1	0.3

^*Unscheduled visit

EDSS, Expanded Disability Status Scale; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, γ-glutamyltransferase; ALP, alkaline phosphatase; CRP, C-reactive protein; MLT, melatonin; nt, not tested; m, months

Discussion

The safety profile of melatonin has been extensively documented in both animal and human studies. Short-term use of melatonin, even at high pharmacological doses, has been consistently shown to be well tolerated, with no serious adverse events reported. Similarly, randomised clinical trials have shown that long-term administration of melatonin is only associated with mild adverse effects that are comparable in frequency and severity to those reported with a placebo [17, 19]. Although large-scale randomised clinical trials that evaluate chronic high-dose melatonin administration remain limited, existing studies confirm its low toxicity and favourable safety profile [18, 20]. In this regard, melatonin has been administered in a wide range of daily doses in humans, ranging from 0.1 mg to 3 g per day (summarised in Supplementary Table S1), with no evidence of serious adverse effects [17–19]. Oral doses of up to 0.3 mg maintain plasma melatonin levels within the endogenous range [26], serving as the threshold between high physiological and low pharmacological doses. In Europe, melatonin is available as a dietary supplement at doses below 2 mg, which can increase endogenous levels up to tenfold without reported adverse effects [27].

Regarding high-dose trials, a pilot study in three patients with amyotrophic lateral sclerosis (ALS) who received oral doses of 30–60 mg/day for 13 months reported no intolerance, side effects, fatigue or changes in biochemical markers [28]. A larger trial involving 31 patients with ALS treated with 300 mg/day of rectal melatonin for 2 years found that serum melatonin levels remained stable over time, indicating that its metabolism was not altered. Routine laboratory tests showed no significant abnormalities [29]. Furthermore, a randomised, double-blind, placebo-controlled trial conducted in 24 male athletes receiving 100 mg/day of oral melatonin or a placebo revealed no evidence of hypertransaminasemia, as demonstrated by normal plasma levels of AST and ALT that remained unchanged throughout the 4-week treatment. In fact, LDH levels decreased significantly after melatonin treatment, with no changes observed in the placebo group [30]. In addition, in a placebo-controlled study conducted in 54 male patients, who had been subjected to varicocelectomy, receiving 400 mg/day of oral melatonin or placebo for 3 months, no adverse effects were reported [31]. Studies in healthy women have also confirmed the safety of high-dose melatonin. In a trial, 12 women who received 300 mg/day for 4 months did not experience adverse events [32]. A double-blind, randomised, placebo-controlled trial in 50 patients (68,18% women) with type 2 diabetes mellitus (T2DM), who ingested 250 mg of melatonin once a day for 8 weeks concluded that melatonin supplementation may be effective in controlling clinical events related to T2DM without reporting adverse events or side effects [33]. In addition, a case report described an improvement in the EDSS score in a woman patient with PP-MS taking 300 mg/day for 4 years, with no reported adverse events [22]. A 4 weeks ingestion of 1 g/day melatonin (4 doses of 250 mg throughout the day) in five human subjects with hyperpigmented skin (4 of them were women) did not show any sign of toxicity related to the eyes, liver, kidneys and bone marrow, beyond increased drowsiness [34]. A randomized controlled trial in 20 women with amenorrhea treated with 3 g/day of melatonin combined with myo-inositol for 6 months found no side effects compared with the control group [35].

Studies also indicate that daily administration of melatonin at low-to-moderate pharmacological doses (20–100 mg) does not cause significant side effects beyond mild sleep disturbances and fatigue. Regarding MS, a recent systematic review has shown that melatonin supplementation (ranging from 2 to 25 mg/day in treatments from 2 weeks up to 6 months) exerts benefits in oxidative stress, inflammatory status and MS symptoms, such as fatigue, without significant safety issues [36]. In particular, in a double-bind, randomised, placebo-controlled trial conducted in 36 patients with RR-MS (26 women) receiving 25 mg/day of oral melatonin or placebo for 6 months, no significant differences were observed in side effects rates compared with placebo [37].

Despite the well-documented safety profile of melatonin, the MELATOMS-1 trial was temporally interrupted and unblinded due to three cases of hypertransaminasemia, all of which occurred in the melatonin group. Given the extensive literature supporting the hepatoprotective role of melatonin [38, 39], this unexpected finding led to a further analysis of patient characteristics and hepatic metabolism in the context of polymedication to explore the possible causal mechanisms underlying the observed increase in transaminase levels.

Hepatotoxicity is an important reason why drugs in development may not continue. Because all participants in the MELATOMS-1 trial received ocrelizumab as background therapy, its potential contribution to the observed hepatotoxicity was carefully considered. Ocrelizumab, a humanized anti-CD20 monoclonal antibody, has a generally favourable liver safety profile. Large clinical trials and long-term studies have not demonstrated drug-induced hepatotoxicity or clinically relevant elevations in liver enzymes in most patients with multiple sclerosis [40, 41]. Most hepatic adverse events are mild and transient [42, 43], while rare cases of acute or autoimmune hepatitis and fulminant liver failure appear to be idiosyncratic and unpredictable [44, 45]. The main liver risk associated with ocrelizumab is hepatitis B virus reactivation [46, 47]; however, all participants in this study were screened negative for HBV infection before enrolment, and no viral reactivation occurred during follow-up. Therefore, ocrelizumab is unlikely to explain the transaminase elevations observed in participants treated with melatonin.

Identifying the exact cause of DILI is often complex [48], but in this study, a temporal relationship with melatonin treatment appears plausible, as hypertransaminasemia occurred exclusively in the melatonin group, and liver enzyme levels improved after discontinuation of melatonin. In addition, case 2 experienced a recurrence of hypertransaminasemia after melatonin reintroduction. However, this patient experienced a sharp decrease in transaminase levels just 1 day after discontinuing melatonin (ALT: 75%, AST: 60%, GGT: 25%), suggesting that other factors, such as a viral infection or gallstone disease, may have contributed.

Unfortunately, no complementary tests were performed to confirm this hypothesis. According to the severity rating scale of the International DILI Expert Working Group, only case 2 was classified as grade 2 (moderate toxicity), while the other two cases were grade 1 (mild). The Roussel Uclaf Causality Assessment Method (RUCAM) score [49] was not calculated due to its limitations in the context of clinical trials.

The three patients who developed hypertransaminasemia were women, a factor linked to an increased susceptibility to drug-induced hepatotoxicity. Epidemiological studies suggest that women have a greater predisposition to develop DILI and acute liver failure than men [50]. This sex-based vulnerability is likely related to differences in drug bioavailability, metabolism and excretion, as well as hormonal influences on liver enzyme activity [51]. Interestingly, the only patient in the melatonin group who did not develop hypertransaminasemia was a man, despite receiving the same dose of melatonin and being the longest-treated participant (14 months of uninterrupted melatonin intake). This observation further supports the hypothesis that sex differences may play a role in susceptibility to hypertransaminasemia.

Another common feature observed in affected patients was a mild increase in CRP levels, which occurred concomitantly with hypertransaminasemia in all three cases treated with melatonin. This consistent temporal association suggests that CRP elevation most likely reflected a secondary acute-phase response to hepatic inflammation rather than a marker of systemic disease activity. Although elevated CRP levels are rare in clinically stable primary progressive multiple sclerosis (PP-MS) [52], mild and transient increases have been reported in association with tissue injury or intercurrent inflammation [53, 54]. In our study, CRP levels remained below 13 mg/L and normalised after discontinuation of melatonin, paralleling the resolution of liver enzyme abnormalities. All participants received ocrelizumab, which is not known to induce liver inflammation or acute-phase responses, and the placebo subject who showed a mild CRP increase (7.1 mg/L) did not show any biochemical or clinical evidence of hepatotoxicity. Melatonin and placebo capsules contained identical excipients, excluding a formulation-related effect. In this regard, the excipient contained in each capsule was microcrystalline cellulose, colloidal silicon dioxide and magnesium stearate. There is no published evidence linking the administration of any of these components, commonly used as components of formulations in clinical trials, with elevated CRP levels or liver injury, when administered alone or in combination with other drugs [55–57]. Altogether, these findings suggest that CRP elevation represented a non-specific acute-phase reaction secondary to mild hepatocellular injury, rather than a direct consequence of melatonin or an exacerbation of PP-MS. However, the lack of CRP measurements at certain time points prevented us from drawing definitive conclusions.

A key finding in this study is that all affected patients were taking multiple concomitant medications that share liver metabolic pathways with melatonin, suggesting a possible mechanism of metabolic overload or enzymatic inhibition. The liver plays a central role in drug metabolism, regulating the clearance of both xenobiotics and certain endogenous molecules. Hepatic metabolism is largely mediated by the cytochrome P450 (CYP) enzyme system, which consists of a wide variety of isoforms, with CYP 1A2, 2D6, 2C9, 2C19 and 3A4 being responsible for 60% of drug metabolism [58]. The main catabolic pathway of melatonin involves the hepatic CYP1A2 enzymatic isoform, which catalyses the conversion of melatonin to 6-hydroxymelatonin (6-OHM) and, to a lesser extent, to N-acetylserotonin (NAS) by O-demethylation (Fig. 3) [59]. These metabolites are subsequently conjugated with sulfate or glucuronide through sulfotransferases (SULTs) and UDP-glucuronosyltransferases (UGTs), and are ultimately excreted in the urine [60]. Furthermore, other CYP isoforms, such as CYP2C9, CYP1C19 and CYP1A1, are also involved in melatonin catabolism, although their activity is considered minor [61].

Fig. 3.

Diagram of the main pathways involved in the hepatic catabolism of melatonin. The major process of MLT breakdown occurs in the liver via the hepatic enzyme CYP1A2, which performs 6-hydroxylation to convert melatonin to 6-hydroxymelatonin and O-demethylation to produce N-acetylserotonin within hepatocytes. Subsequently, both 6-hydroxymelatonin and N-acetylserotonin undergo conjugation with sulfate or glucuronide by sulfotransferases and UDP-glucuronosyltransferases, respectively, and are ultimately eliminated from the body through the urine. In addition, other CYP isoforms, such as CYP2C9, CYP1C19 and CYP1A1, are involved in melatonin catabolism, although their contribution is minimal. CYP, cytochrome P450; UGT, UDP-glucuronosyltransferase; SULT, sulfotransferase

In the MELATOMS-1 clinical trial, melatonin was administered at a high pharmacological dose to ensure its maximum concentration in the central nervous system (CNS) and enhance its potential neuroprotective effects. Many of the drugs taken concomitantly by study participants share the same metabolic pathways as melatonin, including CYP isoforms, SULTs and UGTs. The enzymatic activity of CYPs can be inhibited by certain drugs and/or overwhelmed by competitive substrates, leading to drug accumulation and potential toxicity. This raises the possibility that melatonin may have saturated the enzymatic capacity of CYPs (1A2, 2C19, 2C9 and/or 1A1) as well as the conjugation pathways of SULTs (1A1, 1A2, 1A3, 1B1 or 1E1) [62] and UGTs (UGT1A9 and UGT1A10) [63]. Such saturation could result in the accumulation of hepatotoxic substrates, particularly at inappropriate doses [64].

Previous studies have established a dose-independent association between drugs metabolised by CYP enzymes and an increased risk of DILI [65]. Furthermore, research has shown that drugs inhibiting non-CYP enzymes, such as UGTs, are significantly associated with a higher risk of hepatotoxicity [66]. Since UGTs are involved in the conjugation of endogenous molecules such as bilirubin, bile acids, serotonin, steroid hormones and eicosanoids [67], reduced UGT activity can alter the normal metabolic clearance of these substances, leading to bioaccumulation and prolonged biological effects.

Figure 4 summarises the metabolic pathways involved in the hepatic clearance of medications taken by patients receiving melatonin or placebo. In case 1, the patient was taking acetaminophen, metamizole and dalfampridine, in addition to melatonin. Acetaminophen is excreted primarily in urine as non-toxic glucuronidated and sulfated metabolites [68], but a small fraction undergoes oxidation mediated by CYP (2E1 and to a lesser extent by 1A2 and 3A4), generating N-acetyl-p-benzoquinone imine (NAPQI), a highly reactive metabolite detoxified by conjugation with reduced glutathione (GSH) [69]. At high doses of acetaminophen, the glucuronidation and sulfation pathways become saturated, increasing CYP metabolism and NAPQI production [70]. If excess NAPQI accumulates beyond the available detoxification capacity of GSH, liver necrosis can occur. Although the acetaminophen dose in this case was low, the presence of elevated melatonin metabolites (6-OHM and NAS) may have competed for the glucuronidation and sulfation pathways, reducing acetaminophen clearance and facilitating NAPQI accumulation and liver injury. In addition, metamizole, a known CYP3A4 inducer, may have further increased NAPQI production, compounding the risk of hepatotoxicity (Fig. 5) [71].

Fig. 4.

Enzymes involved in the liver metabolism of drugs taken by patients. The CYP450 isoforms involved and not involved in melatonin (MLT) hepatic metabolism are listed in the left and right columns, respectively. The LiverTox index of each drug is indicated in brackets [64]. Drugs that do not undergo liver catabolism have not been identified. Within phase I reactions, the main CYP isoforms used by each drug are coloured red, and the isoforms used in a minority way are coloured blue. For phase II enzymes, we used the green colour [69]. CYP, cytochrome P450; n/a: not available; UGT, UDP-glucuronosyltransferase; SULT, sulfotransferase

Fig. 5.

Hypothetical scheme of possible drug interactions that would have occurred in case 1. High doses of melatonin would saturate the enzymes responsible for hepatic conjugation with glucuronide and sulfate, UDP-glucuronosyltransferases and sulfotransferases, compromising the elimination of acetaminophen and thus causing an accumulation of NAPQI. CYP, cytochrome P450; NAPQI, N-acetyl-p-benzoquinone imine; UGT, UDP-glucuronosyltransferase; SULT, sulfotransferase

In case 2, the patient was taking dalfampridine, omeprazole, ibuprofen and sertraline, all of which interact with CYP enzymes. Omeprazole and ibuprofen are classified by LiverTox as categories B and A, respectively [64]. LiverTox is a widely used reference tool for evaluating hepatotoxic potential. It is an expert-reviewed online database created by the US National Institute of Diabetes and Digestive and Kidney Diseases and the National Library of Medicine. It provides comprehensive, evidence-based information on drug-induced liver injury (DILI), including clinical features, mechanisms of hepatotoxicity, case reports and references to primary literature (https://www.ncbi.nlm.nih.gov/books/NBK547852/). Omeprazole is metabolised mainly by CYP2C19, whereas ibuprofen is metabolised by CYP2C9 [72]. When melatonin saturates CYP1A2, its metabolism can be diverted to CYP2C19 and CYP2C9, leading to enzyme saturation and reduced clearance of omeprazole and/or ibuprofen, which could contribute to hepatotoxicity (Fig. 6). Although sertraline (LiverTox score of B) is mainly metabolized by CYP2B6 [69], it may have played a role in competing for CYP1A2, 2C9 and 2C19 [73], leading to the subsequent metabolic overflow of omeprazole and/or ibuprofen.

Fig. 6.

Hypothetical scheme of possible drug interactions that would have occurred in case 2. Melatonin competes with other substrates for binding sites to CYP2C19 and CYP2C9 enzymes, increasing the duration of liver exposure to these high liver toxicity drugs. The LiverTox index of each drug is indicated in brackets [64]. CYP, cytochrome P450

Case 3 was taking melatonin, tizanidine, nabiximol, acetaminophen, omeprazole and acetylsalicylic acid (ASA). Tizanidine, as melatonin, is almost exclusively metabolised by CYP1A2, making it a direct competitor for enzymatic processing. Although tizanidine (LiverTox score: C) rarely causes hepatotoxicity, its presence could have contributed to CYP1A2 saturation, along with nabiximol and acetaminophen, both minor substrates of CYP1A2. This could have redirected the melatonin metabolism to CYP2C19 and CYP2C9, leading to the accumulation of omeprazole and ASA, both classified as LiverTox scores: B and A, respectively (Fig. 7) [64]. Furthermore, as in case 1, excess NAPQI production from acetaminophen due to saturation of the glucuronidation and sulfation pathways may also have played a role in liver injury.

Fig. 7.

Hypothetical scheme of possible drug interactions that would have occurred in case 3. Melatonin promotes drug accumulation, some of which could be hepatotoxic, by competing as a substrate for the binding site of the different isoforms of CYP450 involved in liver catabolism in phase I reactions. On the other hand, the glucuronidation and sulfation pathways are also saturated by high melatonin levels, and the CYP systems metabolize more acetaminophen. This leads to increased NAPQI production and GSH depletion, increasing liver damage. The LiverTox index of each drug is indicated in brackets [64]. CYP, cytochrome P450; NAPQI, N-acetyl-p-benzoquinone imine; UGT, UDP-glucuronosyltransferase; SULT, sulfotransferase

On the contrary, the patient who was enrolled in the study for the longest period (Mel 4), did not exhibit any signs of hypertransaminasemia throughout the 14-month treatment period, as confirmed by three biochemical evaluations (Table 3).

Interestingly, this patient had a greater than 50% reduction in plasma levels of ALT and AST levels after 12 months of melatonin treatment, consistent with previous reports on its potential hepatoprotective effects [38, 39]. Remarkably, this patient was the only one whose medications did not share metabolic pathways with melatonin (Fig. 4). Furthermore, the drugs that he was taking had low LiverTox scores: tamsulosin (D), dalfampridine (E) and hydroferol (E), suggesting that melatonin alone is unlikely to induce hepatotoxicity unless other hepatotoxic drugs accumulate.

In line with this interpretation, previous studies evaluating high-dose melatonin in other clinical contexts have not reported hepatotoxicity, even when the compound was co-administered with various medications. In ALS, melatonin (30–300 mg/day) was administered together with riluzole, amitriptyline, baclofen and antioxidant vitamins C and E [28, 29]. Among these, only riluzole shares hepatic metabolic pathways with melatonin, mainly involving the CYP1A2 and UGT enzymes [69] . Mild and reversible elevations in liver enzymes observed in two patients were attributed to riluzole rather than to melatonin itself. The other agents (vitamins C and E, baclofen, magnesium) are metabolized through independent, non-CYP pathways and are not known to interfere with melatonin metabolism. In a RR-MS trial [37], melatonin (25 mg/day) was co-administered with interferon β-1b and glatiramer acetate, and occasionally clonazepam or fluoxetine. None of these drugs are major substrates of the main metabolic routes of melatonin could affect minor secondary pathways. In a PP-MS case report [22], melatonin (up to 300 mg/day) was introduced after discontinuation of corticosteroid, without concomitant immunotherapy. In T2DM, melatonin (250 mg/day) was administered along with standard oral antidiabetic agents, while insulin, immunosuppressants, and anti-inflammatory drugs were exclusion criteria [33].

Taken together, these findings highlight that previous studies differed substantially from our study, in which participants were polymedicated with ocrelizumab and several drugs metabolised through the CYP1A2, CYP2C9, CYP2C19, CYP1A1, SULT and UGT pathways, conditions that may have favored drug–drug interactions and hepatic metabolic overload. Although our findings suggest that such interactions could underline the observed hepatotoxicity, the possibility of DILI associated with melatonin via alternative mechanisms cannot be entirely ruled out. Susceptibility to DILI is highly individual and is influenced by multiple factors, including sex, genetic predisposition, disease state, diet, lifestyle and environment [74].

Although the strength of this study lies in the identification of a significant adverse event, which requires careful consideration for the formulation of future clinical trials involving high-dose melatonin, especially in polymedicated populations, it also has a few limitations, such as the small sample size and the early discontinuation of the trial limit the generalisability of our findings and prevent establishing a definitive dose–response relationship. The heterogeneity of the concomitant medications, together with the absence of detailed pharmacokinetic and pharmacogenetic data, restricted our ability to fully characterise potential drug–drug interactions. Complementary evaluations, including viral serologies, autoimmune markers or imaging studies, were not performed systematically during episodes of transaminase elevation, preventing the definitive exclusion of alternative causes of liver injury. Finally, although the trial was designed to quantify urinary 6-sulfatoxymelatonin (the main urinary metabolite of melatonin) at baseline and every 6 months, the development of hypertransaminasemia in patients prior to the initial 6-month follow-up limited our ability to correlate melatonin levels with the liver effects.

Conclusions

Although melatonin is considered to have a good safety profile, the cases of hypertransaminasemia described in the present study show that high doses of melatonin can, in rare cases, lead to liver enzyme elevations, particularly in patients receiving multiple drugs metabolised through overlapping liver pathways. Although these conclusions should be interpreted with caution due to few limitations, the study reveals a critical safety event with significant implications for the formulation of future clinical protocols involving high doses of melatonin, particularly in populations subjected to polypharmacy. As the use of melatonin expands beyond its conventional role as a sleep aid, these findings underscore the importance of monitoring liver function and evaluating pharmacokinetic interactions when administering melatonin as an adjunctive therapy. Future controlled studies with larger cohorts, incorporating comprehensive pharmacogenetic profiling and detailed metabolic characterisation, are warranted to confirm these observations and define a safe therapeutic window. Defining these parameters is critical to optimise the clinical use of melatonin in CNS and other disorders while minimising the risk of liver adverse effects.

Supplementary Information

Below is the link to the electronic supplementary material.

Funding

Funding for open access publishing: Universidad de Sevilla/CBUA. This work was supported by the Andalusian Government Ministry of Health (PC-0019-2017, PC-0413-2017, PC-0210-2017, PI-0015-2018), the PAIDI Program from the Andalusian Government (CTS-160), and Regional Ministry of Economy and Knowledge of Andalusia (US-1263804) into the European Regional Development Fund Operational Programme 2014 to 2020. E.P.E. and I.B. were supported by the VI Program of Inner Initiative for Research and Transfer of the University of Seville [VI PPIT-US]. A.I.A.L. was supported by the grant US-1263804. Open access fee was funded by the Univesity of Seville.

Declarations

Reporting Guidelines

This study was reported in accordance with the CONSORT guidelines for randomized clinical trials [1].

Conflicts of Interest

The authors have declared that there are no competing interests.

Availability of Data and Material

The datasets analysed during the current study are available from the corresponding authors on reasonable request. Data supporting the findings of this study are deposited in the University of Seville research data repository https://idus.us.es/browse/department?scope=0e8973be-60fe-4985-82cd-13e125b5f5b7&value=Bioqu%C3%ADmica%20M%C3%A9dica%20y%20Biolog%C3%ADa%20Molecular%20e%20Inmunolog%C3%ADa.

Ethics and Regulatory Agency Approvals

This study was conducted in accordance with the principles of the Declaration of Helsinki, Good Clinical Practice (GCP) guidelines, and applicable local regulatory requirements. The protocol was authorized by the Ethics Committee (CEIm Provincial de Sevilla) and the Spanish Regulatory Agency (AEMPS) on the 18 February 2019. Patient data were processed in compliance with European Union General Data Protection Regulation 2016/679 and Spanish Organic Law 3/2018 on the Protection of Personal Data and Guarantee of Digital Rights.

Consent to Participate

Written informed consent was obtained from all participants prior to enrolment.

Consent for Publication

Not applicable.

Code Availability

Not applicable.

Author Contributions

Conceived the idea: Antonio Carrillo-Vico and Patricia Judith Lardone. Data collection: Silvia Jiménez-Jorge, María Ángeles Lobo-Acosta, Rocío López-Ruiz and Sara Eichau. Data Analysis: Silvia Jiménez-Jorge, Ana Isabel Álvarez-López, Eduardo Ponce-España. Pharmacovigilance: María Ángeles Lobo-Acosta, Clara M Rosso- Fernández. Clinical review of patients: María Ángeles Lobo-Acosta, Javier Ampuero, Manuel Romero-Gómez, Clara M Rosso- Fernández. Drafting of the manuscript: Ignacio Bejarano and Patricia Judith Lardone. Obtained funding: Patricia Judith Lardone, Clara María Rosso-Fernández, Juan Luis Ruiz-Peña and Antonio Carrillo-Vico. Administrative and technical support: María Ángeles Géniz, María Isabel García-Sánchez, Manuel Cameán, Vicente Merino-Bohorquez, Silvia Jiménez-Jorge, María Ángeles Lobo-Acosta and Guillermo Izquierdo. Critical revision of the manuscript: Patricia Judith Lardone, Antonio Carrillo-Vico, Ignacio Bejarano, Silvia Jiménez-Jorge, Rocío López- Ruiz, Sara Eichau, Juan Miguel Guerrero, Javier Ampuero, Manuel Romero-Gómez, María Ángeles Lobo-Acosta, Clara María Rosso-Fernández. Approval of the manuscript: all authors.