key: cord-0804871-64o9xfap authors: Boccanegra, Brigida; Verhaart, Ingrid E.C.; Cappellari, Ornella; Vroom, Elizabeth; De Luca, Annamaria title: Safety issues and harmful pharmacological interactions of nutritional supplements in Duchenne muscular dystrophy: considerations for Standard of Care and emerging virus outbreaks date: 2020-05-30 journal: Pharmacol Res DOI: 10.1016/j.phrs.2020.104917 sha: 31b036bd6ae0cfd30574b1b7aa39d7e9f35d8c86 doc_id: 804871 cord_uid: 64o9xfap At the moment, little treatment options are available for Duchenne muscular dystrophy (DMD). The absence of the dystrophin protein leads to a complex cascade of pathogenic events in myofibres, including chronic inflammation and oxidative stress as well as altered metabolism. The attention towards dietary supplements in DMD is rapidly increasing, with the aim to counteract pathology-related alteration in nutrient intake, the consequences of catabolic distress or to enhance the immunological response of patients as nowadays for the COVID-19 pandemic emergency. By definition, supplements do not exert therapeutic actions, although a great confusion may arise in daily life by the improper distinction between supplements and therapeutic compounds. For most supplements, little research has been done and little evidence is available concerning their effects in DMD as well as their preventing actions against infections. Often these are not prescribed by clinicians and patients/caregivers do not discuss the use with their clinical team. Then, little is known about the real extent of supplement use in DMD patients. It is mistakenly assumed that, since compounds are of natural origin, if a supplement is not effective, it will also do no harm. However, supplements can have serious side effects and also have harmful interactions, in terms of reducing efficacy or leading to toxicity, with other therapies. It is therefore pivotal to shed light on this unclear scenario for the sake of patients. This review discusses the supplements mostly used by DMD patients, focusing on their potential toxicity, due to a variety of mechanisms including pharmacodynamic or pharmacokinetic interactions and contaminations, as well as on reports of adverse events. This overview underlines the need for caution in uncontrolled use of dietary supplements in fragile populations such as DMD patients. A culture of appropriate use has to be implemented between clinicians and patients’ groups. Duchenne muscular dystrophy (DMD) is a severe, progressive muscle wasting disorders affecting around 1 in 5 000 newborn boys. First symptoms arise around two to three years of age, after which a progressive loss of muscle tissue leads to wheelchair dependency, usually in the early teens. Hereafter respiratory and cardiac failure occur, which are often the cause of death before the age of thirty [1] . It is caused by mutations in the DMD gene, located on Xp21, which encodes for the dystrophin protein, leading to its absence [2, 3] . A less severe form is Becker muscular dystrophy (BMD), which a short yet partly functional dystrophin protein is expressed [2, 3] . Dystrophin use as pills, tablets, syrups, etc. In USA and EU-countries, dietary supplements are regulated as food, and no prove of safety nor formal approval is required before their commercialization; however, due to the intense and global interest toward these products, there is an increasing attention by regulators with respect to safety issues [37, 38] . For instance, for a new dietary ingredient, the manufacturer must provide information about the reasonable evidence for safe human use of the product. Legally, dietary supplements cannot be claimed to exert any action towards pathology, either for diagnosis, prevention or treatment. Rather, the health claim for a dietary supplement is for correcting nutritional deficiencies that increase the risk of a health-related condition. In parallel, nutrient content claims and structure/function claims are related to the amount of the nutrient and how this has an impact on physiological functions. As described by the Office of Dietary Supplements of NIH (https://ods.od.nih.gov/), although structure/ function claims do not require FDA approval, the label should clearly indicate that the product has not been approved by FDA nor intends to have any drug-related action [37] [38] [39] . Regulators, such as FDA and European Commission (through the European Food Safety Authority; EFSA, https://www.efsa.europa.eu/ en/topics/topic/food-supplements) are active in post-marketing safety surveillance and in providing specific guidance to evaluate sources, and establishing limits for ingredients and contaminants. Consumers, health professionals and industry members can report unwanted reactions to dietary supplements and regulators can take actions to protect the public from unsafe products, including withdrawal due to proven or suspected harmful effects and toxicity. Reasons for recalls include contamination by microbiological species, pesticides, and heavy metals as well as abnormal presence of dietary ingredients with respect to what claimed in the label, including absence, reduction or excess, or the presence of other active principles [40] . In fact, many supplements contain ingredients that have strong biological effects, and such products may not be safe in all people, especially if an underlying pathology or an acute viral or bacterial infection requires concomitant use of other drugs, increasing the risk of potential harmful interactions (see Directive 2002/46/EC [41] ). It should be underlined that food and dietary supplements are also freely available on the web, which increases the risk of consumers being exposed to products of limited quality in terms of content of active principles and contaminants. A great interest has been raised toward dietary supplements in DMD [42] . One of the main reasons resides in the possibility to get adjuvant control of secondary pathogenic events, possibly related to altered metabolism, diet intake or dietary requirements, and based on their claimed mechanism of action. Then, supplements and nutraceuticals can potentially control inflammation and oxidative stress, provide a clue to overcome impaired protein synthesis/proteolysis related to acute necrosis or act as energizer or metabolic booster at skeletal muscle level [42] . The actions of some supplements in DMD have been supported by well-designed preclinical studies, while for many others the action is elusive, and more evidence is needed [28] . Based on what is stated in paragraph 3, any reference to a "therapeutic" action of food supplements without a clear link to dietary deficiency or metabolic requirement, would be misleading. Some of the nutritional supplements could be important in case of a clear nutritional deficit, as may occur in specific phases of the pathology. In this respect, if a dietary deficiency is corrected with a supplement, then an adjuvant therapeutic-like action can be expected. It is anticipated that this would, in any case, be the most reasonable use of any dietary supplement although not always easy to proof preclinically in animal models [37] . The dark side of nutritional supplementation is related to the wrong assumption that these compounds, because of natural origin and normally present in diet, are safe and pivotal to maintain the wellbeing state and potentially help to prevent other pathological conditions, as those related to infections as in case of pandemic COVID-19. This holistic view is totally misleading, since the basis for the action of these exogenous compounds, especially at the high doses chronically used as supplements, follows the same mechanisms that underlie efficacy and safety issues of regular drugs. As anticipated supplements may contain natural active principles that have drug-like effects, like steroids present in a Chinese supplement with claimed beneficial effects in DMD boys [43] , or statins Pharmacological Research 158 (2020) 104917 present in red yeast rice [44] . In this case, an active principle of natural origin can have therapeutic action, i.e. impact on pathology-related events, and act as a disease modifier. However, the active principle should be considered per se as a drug, with no specific reference to dietary requirement and follow the same rule and experimental validation needed for pharmaceutical compounds [34] . For most natural compounds the boundary is very narrow, with doses and underlying rationale making the big differences. Consumers and patients need to be properly informed. Other than being wrong, the assumption of natural-being-safe is dangerous, because caregivers can decide in an autonomous way and without medical prescription or suggestion to implement a supplement in the diet of a DMD boy with the idea that this is without danger and can only help. To increase the level of complexity, legislation can differ in various countries worldwide, with some preparations being considered somewhere "dietary supplements" and "pharmaceutical products" elsewhere. In a global world, as that of rare disease, this can increase confusion and misuse [37] . Unfortunately, this attitude exposes patients to high risk of ADR, may delay or counteract the effect of other therapeutics, and also adds economic burden to families, due to both the high costs of this class of compounds and the poor control of pathology progression. A wide range of dietary supplements is used by DMD patients. The present review is not aimed at entering into the specific rationale of their use; however, in Fig. 2 their main site of action is indicated, while concepts about the mechanism of action and an overview of the preclinical and clinical studies performed in DMD is given in Table 1 . As general considerations, a variety of both preclinical and clinical experimental approaches have been used and many contradictory results have been obtained over the years. Variables including age, stage of the disease, dosage, route of administration, time of exposure and readouts, have to be considered when reviewing efficacy data of these compounds or when designing preclinical and clinical studies. Importantly, the scientific community increasingly recognizes the need of properly conducting preclinical studies in this field, also for de-risking the possible clinical assessment of supplement need in DMD patients [45] , similarly to the standardization in drug preclinical studies [46] . When testing dietary supplements different nutritional requirements between species should also be considered and accurately balanced with the more limited information about doses/levels needed to correct an alteration, so to reach the proper amount in target tissues and have a biological impact, further complicating the general picture. Based on the above consideration, we will presently focus on the main mechanisms that can account for possible safety concerns or harmful interactions for the most used dietary supplements in DMD (Table 1 ) as well as describing specific cases of reported toxicity by pharmacovigilance monitoring in the wide population (Table 2) . The anabolic effect of branched-chain amino acids (BCAAs; leucine, isoleucine, and valine) on animal and human muscles has been well described in several studies [47, 48] and is primarily due to BCAAs' capability to affect pathways involved in protein synthesis (mTOR, S6K1). BCAAs are considered essential amino acids (EAAs) for humans, and, like the other six EAAs, they need to be necessarily introduced through the diet. However, recently, doubts were raised about the real ability of BCAAs to stimulate muscle anabolism, occurring when the rate of muscle protein synthesis exceeds the muscle protein breakdown and depends on the presence of an adequate amount of intracellular EAAs. The intake of BCAAs alone cannot prevent, in the post-absorptive state, the protein breakdown required for the production of the remaining six EAAs, making it impossible to create an anabolic state [49] . In addition, contradictory outcomes have been described in clinical studies [50, 51] , reinforcing the need for proper preclinical assessment in animal models in order to obtain more consistent and reliable results that may be useful for translational research on BCAA effects. The most common side effects revealed during the aforementioned trials were gastrointestinal: decreased appetite, anorexia, nausea, and general stomach discomfort [51] . The potential anabolic effect of BCAAs is a costly process that requires a proper availability of energetic substrates: in fact, leucine administration promotes glucose uptake in skeletal muscle and enhances insulin response [52] . Consequently, it must be taken into account that BCAAs have a complex interaction with glucose metabolism. Supplementation might lower blood glucose levels (see Table 2 ), and this may have additive effects in DMD patients treated with antidiabetic drugs [53] . On the other hand, elevated BCAA can be prognostic of insulin resistance [54] ; also, this aspect can be harmful in steroid treated DMD patients that already experience insulin resistance (v. Fig. 3 ). Another hypothesis to consider is the expression of BCAA transporters in skeletal muscle of dystrophic patients. In fact, muscle mass reduction could readdress the distribution of BCAAs towards other tissues, with changes in the amino acid flow. So far, no data seem to be available concerning BCAA levels in DMD patients. The work of Carunchio et al. in 2010 [55] also suggested that excessive use of BCAAs (with a BCAA-enriched diet, 2.5 % valine, isoleucine and leucine in 1:1:1 ratio) in sport athletes caused a major increase in the risk of amyotrophic lateral sclerosis (ALS) due to enhanced hyperexcitability of cortical neurons and excitotoxicity (see commentary by Manuel & Heckman, 2011 [56] ). A risk to alter calcium homeostasis by BCAA has also been proposed, which would be dangerous in DMD subjects. BCAAs are also important for the ammonia detoxification in skeletal muscle, promoting the synthesis of glutamate, which acts as a substrate for glutamine production. On the other hand, an excess of BCAAs supplementation and, consequently, of glutamine synthesis, may increase glutamine breakdown and the production of ammonia in the intestine and kidneys. This condition might lead to the development of hepatic encephalopathy. For these reasons, attention should be given to the dose of BCAAs administrated [57] . A study of 2004 [58] revealed the presence of dehydroepiandrosterone (DHEA) and 4androstenedione not indicated on the label of BCAA supplements. The abuse of anabolic steroids might cause relevant side effects and alters the normal hormonal production in the body [59] , especially in DMD patients already under steroid therapy (v. Fig. 3 ). Another proposed approach to counteract muscle wasting in DMD is enhancing utrophin expression, a protein sharing 80 % homology with dystrophin. Utrophin has a similar function in the maintenance of the dystrophin-glycoprotein complex (DGC) integrity and myofibre structure. A widespread strategy to improve utrophin expression was to enhance the activity of the muscular isoform of nitric oxide synthase (NOS-μ) and NO synthesis by means of NO-donors administration, like L-arginine (or its precursor, L-citrulline [60, 61] ). NO is also critical to support mitochondrial function and biogenesis [62, 63] . Interestingly, a recent study also showed that the levels of L-arginine and its precursor L-citrulline were significantly lower in mdx muscles with respect to wild type ones [64] . A recent study showed that L-homoarginine (one of the direct products of L-Arg metabolism in human) serum levels were B. Boccanegra, et al. Pharmacological Research 158 (2020) 104917 significantly lower in Becker muscular dystrophy patients [61] . This evidence, if confirmed in DMD patients, might give more support to the requirement of L-arginine and L-citrulline supplementation through diet. Although no other clinical trials with L-arginine administration alone or in combination with different drugs in DMD boys are currently published, a wide range of evidence in healthy and unhealthy subjects have been collected [65, 66] (Table 1 ). L-arginine was administered alone or in combination with multiple active ingredients, in various dosage forms and with varying duration of treatment. This marked heterogeneity in experimental procedures makes it difficult to establish the upper level of intake of L-arginine and the accurate evaluation of its safety and real effect. The absence of serious adverse effects related to L-arginine or L-citrulline administration in human trials confirms the safety profile of these amino acids. However, the most common side effects reported were nausea and mild diarrhoea [67, 68] . This is probably due to the increase of local NO synthesis by means of stimulation of intestinal NO synthase (NOS-1) activity, promoted by L-arginine ingestion. As it is well known, NO is strictly related to intestinal function at different levels, including motility, water retention and electrolyte transport. The excess of local NO synthesis might generate an imbalance in the equilibrium between intestinal absorption and secretion, promoting abnormal water loss and, consequently, nutrient depletion [69] . L-arginine contributes to the regulation of hemodynamic. In fact, animal and human studies demonstrated a hypotensive effect of L-arginine administration, not only related to the increase of NO synthesis but also to the enhanced endothelial cell function and the decreased resistance of peripheral blood vessels [70] [71] [72] . L-arginine also induced hyperkalaemia in animals and humans [73] . A recent study suggested that L-arginine and other dibasic amino acids increased plasma potassium levels, promoting the metal ion efflux from liver and pancreas to the extracellular aqueous environment [74] . L-arginine supplementation downregulates the expression, in liver and ileum, of cationic transporter-1 (CAT-1) that is also able to transport other basic and essential amino acids, like lysine and histidine, with a consequent reduction of EAAs plasma levels. This condition should be avoided in DMD patients. Furthermore, it was demonstrated that L-arginine affected the expression of enzymes involved in its metabolism, as such as arginase I, reducing its own bioavailability [75] . Considering these effects on vascular homeostasis and electrolytes balance, uncontrolled administration of L-arginine, particularly in unhealthy subjects, should be avoided. While an improvement of peripheral blood flow may contrast functional ischemia in DMD patients [76] , L-arginine might cause excessive low blood pressure in DMD patients under treatment with antihypertensive drugs and could have a synergistic effect with agents that increase potassium levels, such as beta-and alpha-blockers. In accordance with this assertion, three cases of palpitations, irregular heartbeat, tachycardia and syncope were reported in healthy subjects after taking arginine supplementation [77] (see Table 2 ). No relevant contaminations have been reported for L-arginine. In 2014, an investigation by the FDA discovered N-acetyl-L-leucine instead of L-citrulline in lots of these supplements with subsequent evidence of hyperammonaemia and related health hazards in patients [68] . Dystrophic muscles show a low glutamine concentration and DMD boys exhibit a significant decrease of glutamine bioavailability in the post-absorptive state, with the result that this amino acid should be Table 1 Mechanisms of action and outcome readouts for efficacy in preclinical and/or clinical studies, for dietary supplements most commonly used in DMD. Claimed mechanism of action Preclinical studies in mdx mice: assessed endpoints Increase protein synthesis Modulation of vascular function ↓ Protein degradation [50] Myofibre phenotype No improvements [51] Motor function [206] L-arginine Enhance utrophin expression Utrophin expression [62, 63] In combination with metformin (AMPK activator) [60] : Necrosis [63] ↑ Activity of mitochondrial electron transport chain Respiratory function [63] ↓ Oxidative stress ↑ Clinical scores Glutamine Antioxidant activity Oxidative stress ↑ Insulin secretion [83] Protein breakdown [207] ↓ Protein degradation [82] No effect on muscle function or strength [ Oxidative stress [103] Muscle function [89] Membrane conductivity [89] Cardiac function [104] Muscle strength (in combination with prednisolone) [ Antioxidant and anti-inflammatory activity Muscle damage and CK plasma levels [110, 111] Clinical trial ended but data not available Necrosis [111, 112113] Regeneration [110] Oxidative stress [111, 112113] [208], Muscle force [111, 113] Antioxidant and anti-inflammatory activity Oxidative stress markers [114] Anti-inflammatory activity Necrosis [133] ↓ Insulin resistance Inflammation [133, 134] Trend towards a decrease of muscle loss [135] Vitamin D Restored insufficient 25-hydroxyvitamin D serum levels in DMD boys In combination with calcium-rich diet [210] : Muscle damage [209] ↑ Bone mineral density ↑ Bone mineral content Resveratrol Anti-inflammatory activity Muscle damage (CK/LDH plasma levels) [103] Oxidative stress [103, 150, 151] Inflammation [211] Autophagy/mitophagy [150, 212] Muscle force [103] Utrophin expression [211] Curcumin Anti-inflammatory activity Muscle strength [152, 153] Muscle damage [152, 153] Inflammation markers [152, 153] NF-kappaB activity [152, 153] Antioxidant activity Muscle damage (CK plasma levels) [168, 169] Necrosis [168, 169] Inflammation [169, 170] Oxidative stress [169, 170] βdystroglycan and utrophin expression [172] Coenzyme Q 10 Antioxidant activity ↑ Muscle strength [179] No effect on echocardiographic parameters [ considered "conditionally essential" for DMD patients [78] . Skeletal muscle plays a crucial role in the stock, metabolism and release of glutamine, maintaining approximately 80 % of the entire body amino acid content [79] . According to this point, it is conceivable that the muscle-wasting condition would account for glutamine deficiency, paving the way for a proper rationale to proceed with glutamine supplementation in several muscle pathologies, including DMD. Nonetheless, clinical trials in DMD patients revealed a lack of functional benefits of glutamine supplementation compared to placebo [80, 81] , despite the encouraging results obtained on the reduction of whole-body protein degradation [82] . A vast number of studies in human subjects that investigated the safety profile of glutamine have been published. No evidence of adverse effects appeared, supporting the harmless of glutamine supplementation [67] . The two most common adverse effects reported after L-glutamine consumption are nausea and vomiting (see Table 2 ). Interestingly, a recent study investigated the effect of glutamine on glucose metabolism in DMD boys [83] . Glutamine, in fact, is involved in gluconeogenesis as a source of carbon skeletons via the tricarboxylic acid cycle (TCA) cycle. After 5 h of glutamine infusion, a rapid and transient increase of insulin plasma levels, and a prompt return to normal values when blood glutamine concentration was maintained at basal level were observed. For this reason, DMD patients under insulin therapy should avoid glutamine co-administration. Also, in glutamine supplements contaminations with anabolic steroids (4-estren3,17-dion, 4-androsten3,17-dion) were found (v. Fig. 3 ) [84] . In the last decades, creatine (Cr) has become the most popular dietary supplementation among athletes, both professionals and amateurs. The reason for this widespread popularity of Cr is attributable to the benefits on muscle performance globally observed in the sports field, not only at competitive level. Cr elicits various effects on muscle metabolism, managing energy resources in skeletal muscle cells. Cr and its phosphorylated form phosphocreatine (PCr) are responsible for the ATP shuttling between mitochondria and cytosol. In this way, during physical activity, myofibres have access to a dynamic reserve of energetic substrates, which remains locked within the cell, also thanks to the negative charge of PCr. Moreover, Cr is involved in the increase of muscle fibre size that might be related to an improvement of protein synthesis or a decrease of protein breakdown [85] . Muscles of DMD patients and mdx mice show a marked depletion of Cr and PCr concentration [86] . The administration of creatine in animal models demonstrated an enhancement of muscle strength and oxidative capacity of skeletal muscle cells with a reduction of myofibres necrosis [86] [87] [88] [89] . These beneficial effects arose from an essential property of Cr to reduce the chronic Ca 2+ -overload, a major feature in DMD myofibres. Importantly, it must be taken into account that the effects of Cr supplementation might be strictly related to the disease phase [90] [91] [92] [93] . Despite these promising results, long-term studies would be necessary to assess benefits and potential side effects of Cr, particularly analysing interaction with corticosteroids. Aside from anecdotal cases of gastrointestinal distress and muscle cramps, a side effect that potentially represents a serious hazard for DMD patients is the weight gain observed after Cr supplementation, becoming more significant with prolonged use of the compound [94] . The increase of body mass is mainly due to water retention and this effect might represent a relevant issue for patients with reduced mobility, and cardiac and hemodynamic sufferance, like DMD boys. In literature, high variability in responsiveness to creatine supplementation was described. This variance primarily depends on the muscular content of Cr, that is higher in fast-twitch type II fibres. In accordance with this evidence, muscles consisting mainly of type fibres II are more susceptible to Cr supplementation. As is well known, in dystrophic muscles the fibre type mainly affected by pathology is the fast one and a progressive replacement of type II myofibres with fibrotic tissue is observed. Due to these reasons, a lack of responsiveness to Cr supplementation in DMD patients might be expected [95] . There are serious concerns about the renal safety of Cr. In detail, literature review and adverse events reports (see Table 2 ) mentioned cases of renal dysfunction in subjects with kidney disease history but also in healthy people taking Cr, even at the recommended dose [96, 97] . Furthermore, there are no studies investigating the effect of Cr long-term administration in children/adolescent. Similarly, no clear evidence is available about the interaction of Cr with drugs potentially harmful for the kidney or mainly excreted via kidney, and possibly used on demand in DMD patients, including antibiotics, antivirals, hydroxychloroquine and other drugs of potential help in viral infections as COVID-19 [98] [99] [100] . It must be considered that Cr supplements most commonly traded in supermarkets, drugstores and also on internet, is a synthetic product derived from the reaction between sarcosine and cyanamide. Possible contaminants produced during this process are creatinine, arsenic, dicyandiamide and dihydrotrianzines; all compounds potentially hazardous for human health [85] . For this reason, careful attention should be paid to the source of the product, rather relying on brands prescribed by clinicians, which ensure the traceability of ingredients and which are subject to inspections that guarantee the respect of the good laboratory practices. One of the supplements with the most multitasking potential is certainly taurine, an amino acid found free in mammalian tissues and particularly abundant in excitable ones. In the human body, taurine exerts various actions, ranging from the antioxidant and the anti-inflammatory effect to the regulation of ion channels functions. This plethora of actions explains the potential use of taurine in the adjuvant therapy of some disorders that affect the muscular system, like myotonia, sarcopenia and disuse muscle atrophy [101] . This is particularly true in conditions characterized by altered tissue amount of taurine as it occurs at different extent in the various phases of dystrophic pathology [89, 102] . Preclinical studies in mdx mice showed amelioration of functional parameters and markers of necrosis, oxidative stress and inflammation, being more effective than creatine [89, 101, 103, 104] . Accordingly, taurine appears to be most effective in early phases of muscle and cardiac pathology, and synergic effect effects with glucocorticoids have been described [105] . This profile may envisage the potential usefulness in chronic use. Taurine supplementation might interfere with hemodynamic: a study in male rats demonstrated that acute injection of taurine induced hypotension and vasodilatation whereas a long-term supplementation in drinking water had hypertensive effect [106] . However, a possible interaction with drugs for blood pressure should be considered. Taurine also modulates the induction of cytochrome P450 (CYP) 3A [107] . For this reason, a possible interaction with CYP3A4 inducers such as glucocorticoids might decrease blood levels of steroids and other drugs in DMD patients (v. Fig. 3) . Furthermore, taurine levels in the body are also controlled by its renal transport, a sodium and chloride dependent mechanism. Then taurine may influence diuresis; in turn, altered kidney function may alter taurine equilibrium in the body [101, 108] . Then, the previous considerations raised for creatine about interaction with drugs at kidney level, also apply to taurine. Adolescent and children consumption of energy drinks with caffeine and taurine is raising globally. Some studies observed an increase in systolic blood pressure and heart rate after consumption. In Table 2 was also reported a case of a young subject who shown symptoms like diarrhoea, tremors and speech disorders after energy drinks consumption. Taurine also induced an increase of left ventricular stroke volume by suppressing sympathetic nervous stimulation and influencing calcium uptake in cardiac muscle cells and a long-term exposure may cause hypoglycaemia [109] . None described. Green tea extract (GTE), derived from Camellia sinensis leaves, contains flavonoids catechins (epigallocatechin-3-gallate (EGCG), epicatechin gallate, epicatechins, gallic acid) that have claimed antioxidant properties. For this reason, GTE supplementation was tested in mdx mice to counteract the oxidative stress in dystrophic muscle cells. Preclinical studies demonstrated that GTE improved muscle function and reduced necrosis in muscle tissues of treated animals. Even though encouraging results are available, there is still a lack of consistency among different studies [110] [111] [112] [113] . EGCG is the principal polyphenol antioxidant present in green tea and has main potency in preclinical tests. EGCG was also tested in DMD patients (https://www.clinicaltrials.gov/ct2/show/NCT01183767) but, currently, there are no results available. GTE extract is also a component of Protandim® (LifeVantage Corp., San Diego, CA), an over-the-counter supplement with antioxidant activity [114] . Other herbal components of this supplement are, in detail, Bacopa monniera extract (45 % bacosides), Silybum marianum extract (70 %-80 % silymarin), Withania somnifera (Indian ginseng) powder, and curcumin (95 %) from Curcuma longa. Protandim was firstly tested on healthy human subjects and a reduction of oxidative stress markers was observed. 6-months administration of Protandim in mdx mice confirmed the positive results with reduction of oxidative damage and profibrotic factors. However, no relevant effects on muscle histology and functional parameters were described. The most frequent side effects are due to the presence of caffeine in GTE extracts, and are headache, nervousness, sleep problems, vomiting, diarrhoea, irritability, irregular heartbeat, tremor, heartburn, dizziness, ringing in the ears, convulsions, and confusion [115] (see also Table 2 ). Caffeine has also a diuretic effect [116] and this may exacerbate the urinary calcium loss in DMD patients. Excessive GTE consumption induced iron deficiency anaemia (case report) [117] . High dose of GTE has hepatic adverse effects: in animal models, EGCG induced hepatotoxicity and several case reports showed hepatic adverse events in humans, associated with an increase of livers enzymes [118, 119] (see Table 2 ). GTE has an inhibitory effect on drug-metabolizing enzymes: CYP1A2, 2C9, 2D6, and 3A4 [120] . The concomitant administration of GTE and drugs metabolized by the aforementioned cytochromes should be avoided, including steroids (v. Fig. 3 ), antibiotics and anti-viral drugs. In addition, EGCG is a competitive inhibitor of the proton-coupled folate transporter (PCFT) that mediates the intestinal absorption of folic acid. Green tea intake could, therefore, interfere with the transport of folate across the small intestine and decrease the bioavailability of this essential vitamin [121] . The lack of folate absorption alters cysteine metabolism, inducing hyperhomocysteinemia (HHcy) [122] . HHcy is involved in skeletal muscle malfunction and could exacerbate the muscle degeneration in DMD. The number of GTE-cardiovascular drug interactions reported in humans increased in the last years and now include simvastatin, rosuvastatin, nadolol, sildenafil, tacrolimus and warfarin. However, the list might be longer, as suggested by the results of animal experiments with diltiazem, verapamil and nicardipine. GTE may, on one hand, increase the exposure to some drugs (simvastatin, tacrolimus), and, on the other hand, reduce the exposure to others (rosuvastatin, nadolol) [123] . Green tea consumption might also impair the correct gastrointestinal absorption of antibiotics (i.e. amoxicillin [124] ) and also inhibits Pglycoprotein (P-gp) activity, a transporter that promotes the cellular efflux of a large variety of drugs including steroids and antiretroviral, increasing their potential toxicity (v. Fig. 3 ) [125, 126] . Importantly, glucuronidation is the most important reaction of phase II metabolism of catechins, catalysed by UDP-glucuronosyltransferase (UGT) isoenzymes (1A3, 1A8, 1A9) [127] . DMD patients treated with drugs or other phenol-based nutraceuticals metabolized by UGT may, therefore, incur in unwanted interactions with GTE [128] . For instance, UGT1A9 is also the major enzyme that metabolizes ataluren, a novel drug recently approved for DMD to promote ribosomal read-through for patients with premature stop codon mutations [31, 129] . Its metabolism could be delayed or impaired by GTE, with potential toxic effects. Focusing on Protandim, most common adverse effects are related to allergic reactions to one or more herbal components of the product. Thirty-two per cent of Chinese tea exceeded the national maximum permissible concentration for Pb [130] . Sources of green tea contamination are: pesticides (HCH and DDTs), environmental contaminants (Polycyclic aromatic hydrocarbons (PAHs)), mycotoxins and microorganisms (Aspergillum, Penicillium, Fusarium), toxic elements (mercury (Hg), lead (Pb), arsenic (As), cadmium (Cd), aluminium (Al), chromium (Cr), and nickel (Ni)), radioactive contaminants (in plantations near areas like Chernobyl and Fukushima), and plant growth regulators [131] . Pb is more bioavailable to tea plants growing in highly acidic soils: Japanese tea had the highest Pb contamination whereas Indian teas had the highest percentage of Cd leaching [132] . On 2012, LifeVantage Corporation announces voluntary recall for lots of Protandim due to contamination with small metal fragments in final products. (https://www.in.gov/isdh/files/LifeVantage_ Corporation_Recall.pdf) Inflammation is a hallmark of dystrophic muscles: for this reason, omega-3 fatty acids (ω3FAs) that are claimed to have anti-inflammatory effects, were tested in dystrophic animals. Supplementation with the omega-3 fatty acid eicosapentaenoic acid (EPA) in mdx mice induced a decrease of inflammation, creatine kinase and improved muscle regeneration [133, 134] . Omega-3 long-chain polyunsaturated fatty acids (Ω3LCPUFA; 2.9 g/ day) intake for 6 months reduced insulin resistance in boys with DMD and showed trends towards slowing the progression of muscle loss and decreasing the fat mass [135] . A few side effects have been reported in subjects using ω3FA supplements. Some of these include fishy breath, upset stomach, diarrhoea, and nausea [136] . PUFA are metabolized and often bio-activated by at least four families of CYP, leading to possible PK interaction with a large number of drugs metabolized by CYP enzymes [137] . In addition, PUFA can lead to several PD interactions with drugs acting as anticoagulants and modulating blood homeostasis. An increment of the International Normalized Ratio (INR) in a patient on therapy with warfarin has been reported. It is important to recall that heparins are used empirically in COVID-19 and risk of bleeding in association with omega 3 should be considered. The patient referred to take a high dose of fish oil (2000 mg/d) daily. PUFA, in fact, might interfere with platelet aggregation, with an additive anticoagulant effect [138] . Accordingly, PUFA may have interactions with NSAIDs. A recent study [139] demonstrated that omega-3 fatty acids are capable of increasing the activity of the ubiquitin-proteasome system (UPS). UPS is a protein network responsible for inducing muscle atrophy and, if not inhibited, promotes protein breakdown. Long-term administration of GC inhibits IGF-1/PI-3 K/Akt/mTOR pathway that normally suppresses the UPS activity. For this reason, the co-administration of omega-3-fatty acids and GC might enhance the pro-atrophic effects of steroids in skeletal muscle. The excessive consumption of fish oil may also cause hypervitaminosis (vitamin D and A) [136] . ω3FA sources (fish and fish oils) are often exposed to environmental contaminants/toxins like mercury, polychlorinated biphenyls and dioxins [140] . A case report of 2012 shown an abnormal mercury blood level in a 38 years old man in Canada (see Table 2 ). Fish oil is also susceptible to oxidation and this process contributes to the rancid conversion of the compound, producing the typical "fishy" smell and aftertaste. This issue suggests an inadequate purification of the product by the manufacturer [140] . Fish oils taken as dietary supplements may contribute significantly to daily intakes of organochlorine contaminants due to the presence of pesticides in marine environments [141] . Most common non-animal sources of ω3FA are flaxseed, walnut, echium and algal oil. Other potential plant sources are soybean and canola that were genetically modified to contain higher quantity of ω3FA. However, as no indication about the use and safety of these oils are spread from the food agencies, the consumption of OGM products should be careful [142] . Insufficient 25hydroxyvitamin D (25(OH)D) serum levels are seen in DMD boys. This is mainly due to the chronic corticosteroid use by DMD boys. In addition, reduced sun exposure due to little outdoor activities can play a role. Lowered levels of 25(OH)D contribute to reducing bone mineral density with an increasing probability of fractures of long bones and vertebrae. Vitamin D supplementation is a widespread strategy adopted in DMD boys to counteract corticosteroid-induced osteoporosis and is recommended by the standards of care [5, 143] . Considering that vitamin D insufficiency may have detrimental role in the immune system, its supplementation in DMD patients should not be interrupted during viral pandemic emergencies, such as COVID-19 [144] . However, caution should be taken according to what described in 5.8.1 and for contaminants (see below). Vitamin D is generally considered safe when taken in appropriate amounts but can cause side effects when taken chronically in high doses. Side effects of vitamin D include weakness, fatigue, dizziness, nausea, vomiting, headache, anorexia, and others. Vitamin D assumption can further increase hypercalcemia as frequently reported i.e. in the UK (Table 2 ) and consequently, calcium supplementation needs to monitored and balanced [145, 146] . Excess vitamin D is also associated with symptoms related to hypercalcemia, such as hypertension, anorexia, nausea, and possible kidney damage [30] . 25-hydroxylation of vitamin D 3 is the first step to produce the most common form of circulating vitamin D and occurs in the liver by means of a reaction catalysed by several CYP enzymes, such as CYP27A1 and CYP3A4 [147] . Literature reports occasional cases of drug-drug interactions with drugs metabolized by the same CYP (e.g., anticonvulsants), with an accelerated degradation of vitamin D and consequent osteomalacia [148] . Vitamin D intake is associated with improved absorption of essential inorganic elements (calcium (Ca), magnesium (Mg), copper (Cu), zinc (Zn), iron (Fe), and selenium(Se)) but has also been linked to enhanced uptake of toxic elements such as aluminium (Al), cadmium (ca), cobalt (Co), and lead (Pb) as well as radioactive isotopes including caesium (Cs) and radioactive strontium (Sr). In children, elevated 25(OH)D 3 levels are associated with an increase in blood lead levels [149] . Bioaccumulation of toxic metals, in turn, counteracts the renal synthesis of vitamin D active form. In 2017, FDA released a warning letter about a potential contamination of batches of liquid Vitamin D products with bacteria Burkholderia cepacia (https://www.fda.gov/news-events/pressannouncements/fda-warns-potential-contamination-multiple-brandsdrugs-dietary-supplements). This bacterium causes severe respiratory infections, particularly resistant to antibiotics treatment. Accordingly, contamination with B. capacia could be dangerous for subjects with respiratory distress and for children with immature immune system, like DMD boys. Resveratrol supplementation has been associated with a variety of health benefits in skeletal muscle, such as increased oxidative capacity, decreased protein degradation, and decreased activation and inflammation. For this reason, this compound was tested in dystrophic animals and preclinical studies suggested that resveratrol supplementation may exert amelioration of muscle function and maturation of skeletal muscle cells by reducing oxidative stress [103, 150, 151] . The mechanism of NF-κB inhibition is proper of another compound already tested on mdx mice: curcumin. This curcuminoid, produced by Curcuma longa, was administered i.p. in mdx mice for 10 days [152] . Treated mice showed an amelioration in muscle function and a mitigation of dystrophic symptoms. Nevertheless, a previous study [153] , demonstrated that NF-kappaB activity of mdx mice of three different ages (10 days; 4 and 8 weeks) was resistant to inhibition by oral curcumin supplementation, suggesting that the route of administration might heavily influence the bioavailability of the compound. In vitro studies, [154] showed that resveratrol is an irreversible inhibitor for CYP3A4 and a non-competitive reversible inhibitor for CYP2E1. CYP3A4 activity is important for the metabolism of calcium channel antagonists (e.g. felodipine, nicardipine), immunosuppressants (e.g. cyclosporine, tacrolimus), antihistamines (terfenadine), sildenafil, antiretrovirals; therefore, a possible interaction with drugs should be considered [155] [156] [157] . In addition, resveratrol is metabolized by liver and intestinal UGTs and may cause, like catechins, unwanted interactions with drugs metabolized by the same enzymes [127] . Resveratrol induces autophagy by directly inhibiting mTOR through ATP competition [158] . Since glucocorticoids inhibit mTOR activity [159] , possible additive effects should be taken into account [160] (v. Fig. 3) . Curcumin is generally considered as safe [161] . Studies in human subjects have shown only moderate adverse effects, like bloating, diarrhoea, headache and nausea [162, 163] . None reported. However, careful attention of production chain for commercial resveratrol is advised, like pesticides, heavy metals and other contaminants can be present in grapes, soy, Polygonon cuspidatum and other resveratrol sources [164] . Analyses on curcumin supplements revealed contamination with metanil yellow (C18H14N3NaO3S), a toxic colorant used to adulterate Curcuma longa powder [165] . Furthermore, other compounds commonly used as adulterants are pigments containing lead, with serious concerns about the safety of these products [166, 167] . Because muscles in dystrophic individuals may produce more reactive oxygen species (ROS) and are thought to be more susceptible to oxidative damage, antioxidant supplementation may serve as an adjunct therapy that can be easily incorporated into treatment plans. Nacetylcysteine (NAC) is a potent antioxidant and has been studied as a potential DMD treatment in mdx mice [168] [169] [170] . In detail, NAC is a precursor of the amino acid cysteine that can itself function directly as an antioxidant, and indirectly through glutathione synthesis. Cysteine is also a precursor of taurine synthesis and the increase in muscle taurine content contributes to the amelioration of strength and function of mdx muscle, both in vivo and ex vivo [171, 172] . Importantly, although it has no direct anti-viral actions, NAC has been claimed to have protective effects against pulmonary damages during viral infections in both preclinical and clinical studies due to its antioxidant and mucolytic activities [173] [174] [175] . Then safety issues of NAC as supplement deserve attention, also for the focus it may have in the media, as occurring during the COVID-19 emergency. A clinical trial in cancer patients revealed mostly gastrointestinal side effects, including nausea, vomiting, bloating and diarrhoea [176] ( Table 2) . Paradoxically, long-term administration of NAC in humans increases oxidative stress after an acute muscle injury induced by eccentric exercise, with higher blood levels of markers of inflammation [177] . A preclinical study in mdx mice showed that NAC significantly impaired body weight gain in young growing mice (both mdx and wild type), and reduced liver weight in C57 mice and muscle weight in mdx mice [178] . None described. Coenzyme Q 10 (CoQ 10 ) is a naturally occurring compound that plays a fundamental role in cell bioenergetics as a cofactor in the mitochondrial electron transport chain. CoQ 10 is also a potent antioxidant and may counteract the excessive ROS production in dystrophic myofibres. The compound was tested in different clinical trials in DMD patients with encouraging results: CoQ 10 supplementation ameliorated the impaired myocardial function and physical performance [179] [180] [181] , although no significant improvement in echocardiographic parameters was revealed [182] . Interestingly, idebenone, a synthetic analogue of CoQ10 sharing its antioxidant profile, is at an advanced stage of development for treating cardiorespiratory dysfunction of DMD patients [182] [183] [184] . There are no serious adverse events reported. One patient developed a headache of moderate intensity associated with high blood levels of CoQ 10 . The event resolved with a decrease of the dose [179] . CoQ 10 supplementation may result in a reduction of diastolic blood pressure and might increase the effect of medication with antihypertensive activity [185] . None described. Sources (animal tissues and extracts and whole grains) have to be ensured for quality and purity. Several studies corroborate the view that melatonin might protect B. Boccanegra, et al. Pharmacological Research 158 (2020) 104917 many tissues, including skeletal muscle, from oxidative stress, due to the antioxidant properties of this compound and its metabolites. In mdx mice, muscle of treated animals showed an amelioration of the contractile function of dystrophic myofibres, accompanied by a decrease of CK plasma levels [186] . In DMD patients, melatonin treatment normalizes plasma pro-inflammatory cytokines and nitrosative/oxidative stress, with a reduction of inflammation and muscle wasting [187] . Recently, interest has been raised around melatonin for its anti-inflammatory, immunomodulatory and sedative actions, that may foresee the use of melatonin as adjuvant to contrast the exaggerated immune response finally leading to acute lung injury and acute respiratory distress syndrome in critical care patients with COVID-19 [188] . These may further increase the use of melatonin as supplements by DMD patients as adjuvant in seasonal viral pandemics. Oral melatonin reduces blood coagulation activity with a dose-response relationship [189] . It has been reported that the evening administration of melatonin in hypertensive patients taking the calcium antagonist nifedipine, interfered with the mechanism of cardiovascular regulation, inducing an increase in blood pressure [190] . Melatonin seems to interfere with calcium signalling via calmodulin. This suggests caution should be taken in uncontrolled use of melatonin in hypertensive patients as well as possible interference with antihypertensive drugs. Lastly, it should be underlined that the actual safety of melatonin was not properly assessed, in paediatric population. Doubts were raised over the potential harmful effect of melatonin supplementation on endocrine and reproductive systems in children and adolescents [191] . Further enquiries are needed to evaluate the effects of melatonin in young subjects. A recent study demonstrated that a quarter of melatonin products sold in one city in Canada contained serotonin, some at potentially significant doses [192, 193] . An increment of serotonin levels might cause symptoms like tachycardia, palpitations and hypertension as observed in some cases reported in Table 2 . Reports indicated that the eosinophilia-myalgia syndrome (EMS) was triggered by the consumption of a contaminated batch of the dietary supplement L-tryptophan (L-Trp). Because melatonin is structurally like to L -Trp (both compounds contain the indole ring functionality), a study investigated the nature of the contaminants in commercial preparations of melatonin. Several contaminants in commercial preparations of melatonin were found (formaldehyde-melatonin condensation product and two structural isomers of hydroxymelatonin). Of interest, an EMS-related contaminant in L-Trp referred to as "peak C" has been proposed as a 5hydroxytryptophan isomer. These findings should raise serious questions about the consequences of long-term ingestion of melatonin preparations containing such impurities [194] . Increasing efforts in DMD are devoted toward therapies able to counteract the absence of dystrophin by means of innovative approaches, such as antisense oligonucleotides (ASO) for skipping mutated exons and restoring reading frame, gene editing such as CRISPR/ Cas9, transfer of microdystrophin gene and cell therapies. Two ASOs, eteplirsen and golodirsen, have been recently approved by FDA and others are currently in clinical trials, as well as gene and cell therapies. Little or no data are present about the possible interaction of those innovative therapies with classical drugs and even more with dietary supplements. Below an attempt to underline the main points of interaction susceptibility of these innovative approaches. ASOs approved for DMD are phosphorodiamidate morpholino oligomers (PMO), very stable molecules in biological systems, administered in weekly regimens and mostly eliminated as such by renal excretion. Then approved ASOs do not undergo main metabolism via hepatic microsomes nor are they substrates of main transporters in the body. So, it is generally believed that the level of interaction with drug, food and dietary supplements is low. Both eteplirsen and golodirsen have little if any activity as inhibitor or inducer of cytochromes [195, 196] , golodirsen being a weak inducer of CYP1A2. This cytochrome is involved in the metabolism of various commonly used drugs and also involved in the biotransformation of PUFA; then interaction cannot be excluded. Although the available information does not support major harmful interactions, these compounds have potential toxicity at kidney and liver level, suggesting that these tissues could be a possible site of harmful interactions. Moreover, since they are not very well absorbed at cellular level and they are excreted very fast via the kidney, supplements increasing renal clearance could interfere with their efficacy. Other ASOs under development may have different profiles and specific interaction can occur. Patients in clinical trials should then avoid using dietary supplements without medical control. Cell therapy represents another innovative approach for many diseases, including DMD. Various muscle progenitor cells have attracted interest, with some of them, such as mesangioblasts, tested in clinical settings, due to their ability to cross vessel wall in order to reach the site of the damage [197, 198] . Although very little is known about possible interaction with drugs or with dietary supplements, it is intuitive to predict they may occur at various stages of cell therapy. In fact, muscle precursors are highly proliferating cells which are known to critically depend, for proliferation, fusion and adhesion, on nutrients, such as glucose and amino acids, via high expression of specific transporters such as CD98hc [199, 200] . Also, extravasation may need a concerted action of cytokines and molecules involved in oxidative stress signalling and endothelial permeability, which may also be modulated by either drug or supplements. Another point of interaction can be related to a massive immune response that may be induced by the administration of high amounts of cells, which can also be influenced by supplements such as BCAA [201] . Then, although not easy to decipher, interaction with complex outcome may occur between cell therapies and dietary supplements. The idea behind gene therapy is to treat the pathology by substituting the defective gene(s) or at least one copy of it. For what it concerns muscular dystrophy, considering the size of dystrophin protein (usually not fitting in an adeno-associated virus (AAV)-vector), the transfer of microdystrophin is under clinical assessment [202] . One of the main points of interaction relates to the need to control the immune reaction to viral vectors and the new protein, then immunosuppressive drugs can be required. Accordingly, supplements able to modulate PD or PK profile of immunosuppressive drugs should be used with caution. Another promising approach for DMD is nowadays offered by genome editing using the CRISPR/CAS9 technique. For this approach, important concern can be raised for off-target actions and gene mismatch [203, 204] , which can modify key organ functions for drug and supplement mechanism and clearance. Basically, each of these new therapies presents a peculiar mechanism of action, also because they use different cell machinery and they act at different levels (DNA/RNA/protein). For this reason, it will be pivotal to fully understand and not rule out possible interactions with supplements. During the last years, the interest in natural, dietary supplements is increasing in the DMD field as adjuvant approaches in controlling both dietary requirement and pathological events. The recent emergency due to the outbreak of SARS-CoV-2 further increased the interest toward dietary supplements, among the population worldwide. A recent report of New York Times on March 23, 2020, underlined the increase of sales of supplements with claimed action on cold and flu with respect to the same period of 2019, with specific peaks for so called immune boosters, among which vitamin C, zinc, and others, for the hope to prevent or better manage the Sars-CoV-2 infection by reinforcing natural defences (https://www.nytimes.com/2020/03/23/well/live/coronavirussupplements-herbs-vitamins-colds-flu.html). Similarly, a Google search of "dietary supplements" and "COVID-19" on March 31st, 2020 led to more than 12 million of results, underlying the great interest on this topic from different stakeholders. It is therefore understandable, and even more justified by the fragility of the population, that such interest also arises from DMD community. This for the hope to improve standard of care as well as to get protection in case of an unexpected health emergency, as dramatically arose for COVID-19 recently emerged from World Duchenne Organization (https://www.worldduchenne.org/news/live-covid-19coronavirus-and-duchenne-becker-muscular-dystrophy/). Many caregivers/patients may mistakenly assume these supplements are safe. They think that if a supplement will not be beneficial then it will also not be harmful, so it does not hurt to try. This can be amplified by media and internet, also in relation to economic interests. Consequently, the risks of dietary supplements are seriously underestimated and often their use is not discussed with experts (either medical specialists, practitioners or pharmacists), nor related to any proven nutritional deficits. In fact, most of the dietary supplements discussed herein and even those with claimed effect on the immune system, such as vitamin C, are present in food, especially fresh fruits and vegetables. Then, a proper diet, healthy and varied, is the first aid to supply necessary essential elements, amino acids and vitamins. Supplements can, on the other hand, exert unwanted effects related to the use of excessive doses or interact with other medicines patients are using, either inhibiting or enhancing their efficiency and cause serious health risks. There is often no solid proof of dietary supplements effectiveness, while many side effects have been reported, including the complication or the ineffectiveness of outcome of other therapies (see Table 2 ). Then, prolonged use of supplements may cause several adverse effects, that often lead to emergency department visits and, in severe cases, hospitalization. In United States alone, approximately 23 000 emergency department visits and 2154 hospitalizations are connected every year to side effects related to dietary supplement [205] . For DMD and also for BMD patients these interactions can be of particular relevance as they can occur with drugs used for standard care or with innovative therapies during clinical trials. A similar situation also applies in emergency cases, as in the current pandemic by Sars-CoV-2. In fact the treatment of symptomatic COVID-19 patients is still a largely empirical approach with a wide variety of drugs according to the phase of the disease and ranging from antiviral and antibiotics, repurposed old drugs, such as chloroquine and hydroxychloroquine, to immunosuppressants, monoclonal antibodies and low-molecular weight-heparins. All these class of therapeutics have either a low therapeutic index or can per se at high risk of ADR events which may increase with uncontrolled D-D and D-S associations. In addition, more than 1200 clinical trials are currently ongoing on COVID-19 worldwide (clinicaltrial.gov) which reinforce the need for caution in uncontrolled use of supplements. Another issue is that the quality check of these supplements is not always as rigid as it should be and they can be contaminated, certainly when they are retrieved from less controlled sources, like via the internet. Awareness of the risks of uncontrolled use should be raised amongst caregivers and patients. They should be encouraged to always first discuss with their clinicians and pharmacists before starting supplement use and only retrieve them from certified sources, like their pharmacy. Supplement use can also be a confounder in clinical studies and thereby influence the results. Unexplored interactions may reduce the efficacy of innovative therapies in clinical trials or cause unexpected toxicity. A database about the use of food supplements in DMD and BMD population, also in relation to geographical distribution, would be important, also with the aim to carry on properly conducted clinical studies. In parallel, it is important to consider that there is an increasing awareness to focus on dietary requirement in DMD patients. Then a reinforcement of active surveillance may help to detect ADR by dietary supplements, while patients' registries can be of support for better understanding supplements use in relation to geographic and cultural aspects. Accordingly, it is pivotal to reinforce translational research in the field of supplements, considering the different nutritional requirements in different species used for preclinical studies [28, 29] . This review underlines that supplements may represent an important support in nutritional deficit occurring in DMD patients, although dosage and efficacy need to be better established with controlled studies [28] . Safety is a main concern, based on the concept of "primum non nocere" (first, do no harm). Caution has to be taken in properly discussing with experts before deciding to start using supplements. Also, supplements from unknown sources or acquired via the web or outside pharmacy or other authorized retailers should always be avoided. In this respect, the active role by regulators and health professionals helps to provide correct information, in order to avoid direct use of products that escape the normal paths of distribution or do not provide the possibility to check quality control or surveillance [37] . The uncontrolled use of dietary supplements may, in fact, represent an important confounding factor during the development of new therapies or during studies aimed at repurposing drugs in DMD. The pandemic emergency of COVID-19 provided further evidence of this critical issue, as seen by the greater demand for dietary supplements. Patients and caregivers aim to be protected, especially for the evident difficulties to put in place effective social distance for proper assistance of fragile patients. However, also in these conditions, the best prevention resides in efforts in maintaining proper food, hygiene and individual protection devices for patients and/or caregivers. Although specifically focused on DMD, the considerations raised in the present review can also apply to BMD and to other neuromuscular and non-neuromuscular rare patients, that share similar fragility, complexity and uncertainties. The work has been supported by DPP-NL grant to ADL on preclinical assessment of dietary supplement effects in dystrophinopathies and raised in the frame of a task force on dietary issues and nutrition by World Duchenne Organization (https://www.worldduchenneday.org/ category/nutrition-in-dmd/). The fellowship of DPP-NL to OC for a project on artificial muscle as a new platform for testing the efficacy of novel compounds is also gratefully acknowledged. The contribution of Italian PRIN 2017 grant 2017FJSM9S_005 to ADL is also acknowledged. The authors disclose non-conflict of interest. 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metabolism via UDPglucuronosyltransferase: a barrier to oral bioavailability of phenolics Ataluren pharmacokinetics in healthy japanese and caucasian subjects Scale and causes of lead contamination in Chinese tea Residues and contaminants in tea and tea infusions: a review Monitoring of essential and heavy metals in green tea from different geographical origins Eicosapentaenoic acid decreases TNF-alpha and protects dystrophic muscles of mdx mice from degeneration EPA protects against muscle damage in the mdx mouse model of Duchenne muscular dystrophy by promoting a shift from the M1 to M2 macrophage phenotype Evidence of muscle loss delay and improvement of hyperinsulinemia and insulin resistance in Duchenne muscular dystrophy supplemented with omega-3 fatty acids: a randomized study A systemic review of the roles of n-3 fatty acids in health and disease Cytochrome p450 enzymes in the bioactivation of polyunsaturated Fatty acids and their role in cardiovascular disease Fish oil interaction with warfarin Omega-3 multiple effects increasing glucocorticoid-induced muscle atrophy: autophagic, AMPK and UPS mechanisms Safety considerations with omega-3 fatty acid therapy Organochlorine residues in fish oil dietary supplements: comparison with industrial grade oils Bioavailability and potential uses of vegetarian sources of omega-3 fatty acids: a review of the literature Optimizing bone health in Duchenne muscular dystrophy Vitamin d as an immunomodulator: risks with deficiencies and benefits of supplementation Vitamin D intoxication associated with an over-the-counter supplement High-dose oral vitamin D3 supplementation in rheumatology patients with severe vitamin D3 deficiency Cytochrome P450-mediated metabolism of vitamin d Phenobarbital suppresses vitamin D3 25-hydroxylase expression: a potential new mechanism for drug-induced osteomalacia Vitamin d, essential minerals, and toxic elements: exploring interactions between nutrients and toxicants in clinical medicine Resveratrol decreases oxidative stress by restoring mitophagy and improves the pathophysiology of dystrophin-deficient mdx mice Resveratrol induces expression of the slow, oxidative phenotype in mdx mouse muscle together with enhanced activity of the SIRT1-PGC-1alpha axis Curcumin alleviates dystrophic muscle pathology in mdx mice Progressive nuclear factor-kappaB activation resistant to inhibition by contraction and curcumin in mdx mice Inhibition of CYP3A, CYP1A and CYP2E1 activities by resveratrol and other non volatile red wine components Resveratrol modulates drug-and carcinogenmetabolizing enzymes in a healthy volunteer study Drug interaction potential of resveratrol The role of cytochrome P450 in antiretroviral drug interactions Resveratrol induces autophagy by directly inhibiting mTOR through ATP competition Crosstalk between glucocorticoid receptor and nutritional sensor mTOR in skeletal muscle Muscle inactivation of mTOR causes metabolic and dystrophin defects leading to severe myopathy Potential therapeutic effects of curcumin, the antiinflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases Phase I clinical trial of oral curcumin: biomarkers of systemic activity and compliance Phase IIa clinical trial of curcumin for the prevention of colorectal neoplasia Ultrasonic-assisted extraction of the botanical dietary supplement resveratrol and other constituents of Polygonum cuspidatum Evaluation of turmeric powder adulterated with metanil yellow using FT-Raman and FT-IR spectroscopy Contaminated turmeric is a potential source of lead exposure for children in rural Bangladesh Turmeric means "yellow" in Bengali: lead chromate pigments added to turmeric threaten public health across Bangladesh N-Acetylcysteine treatment of dystrophic mdx mice results in protein thiol modifications and inhibition of exercise induced myofibre necrosis N-acetylcysteine treatment reduces TNF-alpha levels and myonecrosis in diaphragm muscle of mdx mice N-Acetylcysteine ameliorates skeletal muscle pathophysiology in mdx mice Increasing taurine intake and taurine synthesis improves skeletal muscle function in the mdx mouse model for Duchenne muscular dystrophy Effect of N-acetylcysteine plus deferoxamine on oxidative stress and inflammation in dystrophic muscle cells N-acetylcysteine synergizes with oseltamivir in protecting mice from lethal influenza infection Nutraceuticals have potential for boosting the type 1 interferon response to RNA viruses including influenza and coronavirus Attenuation of influenza-like symptomatology and improvement of cell-mediated immunity with long-term N-acetylcysteine treatment Pharmacokinetic and pharmacodynamic studies of Nacetylcysteine, a potential chemopreventive agent during a phase I trial Supplementation with vitamin C and N-acetyl-cysteine increases oxidative stress in humans after an acute muscle injury induced by eccentric exercise, Free Radic Pre-clinical evaluation of N-acetylcysteine reveals side effects in the mdx mouse model of Duchenne muscular dystrophy Cooperative International Neuromuscular Research Group I. CINRG pilot trial of coenzyme Q10 in steroid-treated duchenne muscular dystrophy Two successful double-blind trials with coenzyme Q10 (vitamin Q10) on muscular dystrophies and neurogenic atrophies Biochemical rationale and the cardiac response of patients with muscle disease to therapy with coenzyme Q10 Effectiveness of Coenzyme Q10 on echocardiographic parameters of patients with Duchenne muscular dystrophy Phase III Study of Idebenone in Duchenne Muscular Dystrophy Efficacy of idebenone on respiratory function in patients with Duchenne muscular dystrophy not using glucocorticoids (DELOS): a double-blind randomised placebo-controlled phase 3 trial The effects of coenzyme Q10 supplementation on blood pressures among patients with metabolic diseases: a systematic review and metaanalysis of randomized controlled trials Melatonin improves muscle function of the dystrophic mdx5Cv mouse, a model for Duchenne muscular dystrophy Melatonin treatment normalizes plasma pro-inflammatory cytokines and nitrosative/oxidative stress in patients suffering from Duchenne muscular dystrophy COVID-19: Melatonin as a potential adjuvant treatment Oral melatonin reduces blood coagulation activity: a placebo-controlled study in healthy young men Cardiovascular effects of melatonin in hypertensive patients well controlled by nifedipine: a 24-hour study Potential safety issues in the use of the hormone melatonin in paediatrics Melatonin natural health products and supplements: presence of serotonin and significant variability of melatonin content Poor quality control of over-the-counter melatonin: what they say is often not what you get Contaminants in commercial preparations of melatonin Drug metabolism and pharmacokinetic strategies for oligonucleotide-and mRNA-based drug development In vitro pharmacokinetic evaluation of eteplirsen, SRP-4045, and SRP-4053; three phosphorodiamidate morpholino oligomers (PMO) for the treatment of patients with duchenne muscular dystrophy (DMD) (P5.061) Intra-arterial transplantation of HLAmatched donor mesoangioblasts in Duchenne muscular dystrophy Transplantation of genetically corrected human iPSC-derived progenitors in mice with limb-girdle muscular dystrophy Amino acid transport associated to cluster of differentiation 98 heavy chain (CD98hc) is at the cross-road of oxidative stress and amino acid availability Endothelial progenitor cells physiology and metabolic plasticity in brain angiogenesis and blood-brain barrier modeling Novel metabolic and physiological functions of branched chain amino acids: a review Systemic AAV micro-dystrophin gene therapy for duchenne muscular dystrophy Functional rescue of dystrophin deficiency in mice caused by frameshift mutations using Campylobacter jejuni Cas9 CRISPR for neuromuscular disorders: gene editing and beyond Emergency department visits for adverse events related to dietary supplements Supplementation with a selective amino acid formula ameliorates muscular dystrophy in mdx mice Hankard, l-Glutamine administration reduces oxidized glutathione and MAP kinase signaling in dystrophic muscle of mdx mice Endurance capacity in maturing mdx mice is markedly enhanced by combined voluntary wheel running and green tea extract The effect of vitamin d supplementation on skeletal muscle in the mdx mouse model of duchenne muscular dystrophy Low bone density and bone metabolism alterations in Duchenne muscular dystrophy: response to calcium and vitamin D treatment Resveratrol decreases inflammation and increases utrophin gene expression in the mdx mouse model of Duchenne muscular dystrophy Resveratrol ameliorates mitophagy disturbance and improves cardiac pathophysiology of dystrophin-deficient mdx mice Melatonin treatment counteracts the hyperoxidative status in erythrocytes of patients suffering from Duchenne muscular dystrophy None.