Cardiovascular effects of fingolimod: A review article.

Multiple sclerosis (MS) is a chronic auto-immune disease. Most therapeutic strategies for treatment of this disease direct immune modulation and control of inflammatory processes. First-line therapeutic agents showed moderate efficacy and frequent side-effects with moderate efficacy in trials. Their parental administration and limited long-term adherence restrict their efficacy compared with second-line therapies. Fingolimod as a second-line therapeutic agent has been shown to reduce annualized relapse rate, risk of disability progression and inflammatory activity of relapsing MS. Safety and efficacy FTY720: Safety and efficacy issues are the main metrics for judgment of drug efficacy. In this article, we focus on cardiovascular effects of FTY720 treatment. Effect of FTY720 on rate and rhythm, impact of FTY720 on endothelial cells, its atheroprotective effects, its effects on cardiac transplantation outcomes, vascular complications of FTY720, effects of FTY720 on endocrine functions and interaction of FTY720 with cardioactive agents are explained in this review article.

Introduction
Multiple sclerosis (MS) is considered as a chronic autoimmune disease. 1,2 Therapeutic strategies direct immune modulation and control of inflammatory processes. Until now, five first-line and two second-line therapeutic agents are available. 3 First-line therapies as interferon beta-1 and glatiramer acetate showed moderate efficacy and frequent side-effects with moderate efficacy in trials and due to their parental administration, limited long-term adherence consequently restrict their efficacy compared with second-line therapies as fingolimod and natalizumab. 3,4 Thus, emergence of novel drugs is seriously needed.
Drug development for MS is a challenging field of science.
Fingolimod 2-amino-2-[2-(4-octylphenyl) ethyl]propane diolhydrochloride) also known as FTY720 or Gilenya, is a Food & Drug Administrationapproved agent for treatment of relapsing remitting MS (RRMS). [4][5][6] This compound derived from myriocin, a component isolated from the culture filtrate of natural product ascomycete Isaria sinclairii. 7 This licensed drug offers a far more convenient route of administration. This sphingosine-like synthetic analog sequesters autoreactive thymocytes and lymphocytes from lymph nodes through its similarity to gatekeeper sphingosine-1-phosphate (S1P). This fungal metabolite is oral superagonist of pleiotropic S1P receptor (S1PR), which blocks various signaling pathways mediated by interaction of this endogenous lysophospholipid and its receptor. 8 S1P has five major subtypes (S1P1-5), in which each of them has a special pattern of expression. 9 FTY720 is a non-selective agonist of S1PR. 10 Marked S1P1 receptor internalization occurs upon treatment by FTY720. S1P is crucially involved in vascular barrier function, thus regulate inflammation, coagulation, vascular homeostasis, Iranian Journal of Neurology angiogenesis, tumor metastasis, and atherosclerosis. 11 By blocking S1PR on the surface of lymphocytes and cells in central nervous system, including glial cells and neurons; it reduces annualized relapse rate (ARR), risk of disability progression, tissue damage, and brain atrophy and inflammatory activity of relapsing MS. 12 Pharmacokinetics and Pharmacodynamic Pathway of FTY720 FTY720 is a pro-drug, which is reversibly phosphorylated to its biologically active moiety phospho-FTY720 (FTY720-P) by sphingosine kinase (SphK2) within minutes to construct a nonselective S1PR agonist. 13,14 By CYP4F isoenzyme, FTY720 is irreversibly metabolized to its metabolites. 15 Fingolimod in its phosphorylated form binds to four of the five S1PRs (except S1PR2). 16 It has a high blood bioavailability by its oral administration and has a low inter-individual coefficient of variation. 17 It should be used with caution when combined with class Ia and III anti-arrhythmic agents, beta-blockers, and ketoconazole. 19 Interaction of FTY720 with Cardioactive Agents Interaction between FTY720, atenolol and diltiazem is unlikely. A similar acute negative chronotropic effect is elicited using 5-mg single dose of FTY720 and atenolol alone. Addition of FTY720 to atenolol leads to moderate further reduction of heart rate (15% lower). Addition of a calcium channel blocker to FTY720 was not associated with further lowering of heart rate compared with alone. FTY720 did not alter antihypertensive effects of atenolol. Stronger negative chronotropic response to FTY720 alone (<50 bpm) was parallel with no or further decrease heat rate when combined with atenolol. The negative chronotropic effects of FTY720, was stronger than diltiazem alone. 15 P-glycoprotein activity is reduced by FTY720/FTY720P, which leads to the increase in verapamil and loperamide uptake. 16 Safety and Efficacy FTY720 Safety and efficacy issues are the main metrics for judgment of drug efficacy. Thus, the aim of this review article is to prepare information about the advantages and disadvantages of FTY720 regarding cardiovascular system. In this narrative review article, we have searched articles published on the impacts of FTY720 on cardiovascular system using search keywords of MS, cardiovascular system, FTY720, sideeffects, and complications. FTY720 has been shown to be superior to placebo and INF-1B in phase II trials. 19 In a trial, assessing effects of daily oral therapy in MS (FREEDOMS) and comparing FTY with intramuscular interferon beta-1a administered once weekly [Trial Assessing injectable interferon vs. FTY720 Oral in Rapidly progressing MS (TRANSFORMS) are two phase III studies on FTY720. 20 Among side-effects of fingolimod, fatigue, nasopharyngitis, and influenza have been reported more frequent than others. 21 Other reported sideeffects are as headache, fever, chills, muscle pain, swelling in hands/feet, loss of appetite, susceptibility to infection, higher risk of herpetic infections, itching, mild hair loss, eczema, itchy rash, diarrhea, back pain, cough, dizziness, nausea, vomiting sensitivity to light, numbness, tingling, weight loss, weakness, respiratory disturbances as wheeze and dyspnea, liver enzyme elevation, jaundice clay-colored stool, sores in the throat and mouth, sinusitis, bronchitis, depression, vision problems as blurred vision, and macular edema. 22-25 It passes from breast milk with unknown effects on infants of breastfeeding women. Indeed, FTY720 induce fetal harm as teratogenicity and embryopathy in animal pregnancy (category C). 26 The effects of FTY720 on human pregnancy are not yet known. Thus, women at childbearing age under treatment with fingolimod should apply tight birth control strategies. The safety of FTY720 for pediatric age is not well-demonstrated. In addition to its effects on lymphocyte migration, there is increasing evidence for cardiovascular side-effects of fingolimod. Since the use of FTY720 in clinical practice became limited partly due to concerns for cardiac effects, hereby, we focus on cardiovascular effects of FTY720 treatment. Effects of FTY720 on Rate and Rhythm S1PR agonists regulate cardiovascular functions; therefore, FTY720 targets cardiovascular system. Indeed, S1P is involved in embryonic development of cardiovascular system. 27 In phase I and II clinical trials, fingolimod was known to be a safe agent from cardiovascular aspects, except for acute modest, but significant transient and dose-dependent bradycardia. 28 Symptomatic bradycardia, which occurs in about 0.5% of cases is most often selflimiting. 29 Rarely, occurrence of fatal bradyarrythmia using FTY720 has also been reported. 30 These effects have also been observed in healthy volunteers. 31 The decrease in mean nadir heart rate is up to 10 bpm after first does without incremental decrease in heart rate after day two of drug. 4 Most often, heart rate and conduction normalized in 24 h after first Reduction of heart rate occurs maximally within the first 4-6 h after the first does with consequent attenuation over time by continues consumption.
The magnitude of bradycardia did not increase with repeated dosing despite of increase in its blood concentration 3 and with continued treatment, heart rate returns toward baseline values. 11,15 Following treatment initiation, an approximate 10 bmp decrease in heart rates. In the first 12 h post-dose, a dose-dependent decrease in heart rate was evident, in which the adjusted mean heart rate was as follow: 73.6 bpm for http://ijnl.tums.ac.ir 4 July Hp4: 0.5 mg (adjusted mean treatment difference: 7.9 bpm) and 69.6 bpm for 1.25 mg (adjusted mean treatment difference: 11.9 bpm). Decrease in heart rate was more evident in 5 h and 3 h post-dose, which persisted for 8 h and 24 h dose time interval, heart rate became similar to placebo group. Mean heart rates on the day 7, was lower for both 0.5 and 1.25 mg vs. placebo group throughout the first 24 h dose interval. The heart rates over 12 h post-dose were still approximately 10 bpm group by day 14. This side-effect has been reported to be most often asymptomatic. The mean heart rate is lower after once daily oral FTY720 compared with intravenous administration (53 bpm) due to the higher blood level of active metabolites of fingolimod by former. 15 Fingolimod-induced bradycardia is totally reversible using atropine. Interestingly, an intravenous titration of atropine during fingolimod administration could prevent typical nadir heart rate which occurs around 4 h post-dose of fingolimod.
The mean total dose of 1.4 mg atropine was able to reverse the bradycardia attributed to fingolimod. Continued atropine titration to the mean total of 1.9 mg, was associated with raised and maintained heart rate at lower normal limit lasting for a median 3.5 h. The therapeutic doses of atropine for reversal of FTY720-related bradycardia are within the normal therapeutic range of atropine for the treatment of acute asymptomatic and sinus bradycardia. Acute effects of atropine would occur at the first 24 h after FTY720 administration. 32 FTY720 has been shown to have non-significant effects on circadian rhythm, oxygen exchange, airflow and hemodynamic variables as cardiac output and systemic vascular resistance (SVR) during 14 days treatments in healthy volunteers. 15,34 Benign atrioventricular block (type I or Wenckebach) has reported using FTY720. 32 An approximate, 8-10 ms increase in PR-interval has been reported using FTY720, without any change on QRS or QT intervals Despite of slowing atrioventricular conduction, the incidence of Mobitz type II atrioventricular blocks and 2:1 atrioventricular block is respectively. Conduction abnormalities showed to regress during the time and in therapeutic doses, higher degrees of the block were not seen. 29 S1PRs are not only expressed on lymphocytes, but also expressed on the surface of atrial myocardial cells. All S1PRs (S1P1-3) in heart are stimulated using S1P which leads to activation of (Gi, Gq and G12/13) but only S1P1 and S1P3 receptors are activated using FTY720, which leads to activation of Gi. 30,31 Thus, the underlying mechanism of bradycardia is due to the activation of inwardly rectifying Gà1-proteinregualted potassium channel (GIRK/IKACh) channels in atrial myocytes and endothelial cells). The function of acetylcholine-regulated potassium channel (KACh) is stimulated by S1P. S1PR regulates heart rate through binding to its receptors on the surface of atrial myocytes. 33 This inhibited cardiac pacemaker activity is similar to the vagally-mediated cardiac effects through the same G protein-gated potassium channel with different pathway FTY720 induces dephosphorylation of cTnI in ventricular myocytes. Repeated dosing of fingolimod leads to S1RPs internalization and cessation of signaling. 34

Effects of FTY720 on Endothelial Cells
Due to the tissue-specific arrangement of S1PR subtypes, diverse differential cardiovascular effects are not unexpected. S1P1/3 are the major mediators of S1Prelated actions on cardiac microvascular endothelial cells. 30 Specifically, subtypes of S1P and S1P3 are both expressed on the surface of endothelial cells and are involved in vascular stabilization. 34,35 Identification of S1PR involvement in endothelial cell differentiation for the first time, hints to the occurrence of cardiovascular side effects by fingolimod. 36,37 S1P is participated in endothelial cell proliferation, differentiation, migration, and survival. 30 S1P stimulates functional capacity of endothelial progenitor cells. 38,39 It also affects smooth muscle or bone marrow cells through activation of G protein coupled S1PRs. S1P is involved in maintenance of vascular endothelial barrier integrity and angiogenic homeostasis. 30 Activation of S1P1 promotes angiogenesis, whereas stimulation of S1P3 impairs barrier function. 39 FTY720 acts on endothelial cells and is involved in the preservation of vascular integrity through enhancement of adhesion junctional assembly and endothelial barrier functions. 40 FTY720 might act as an agonist for S1P1 and a functional antagonist for S1P3, in order to keep the balance between angiogenesis and microvascular barrier function. 30 The agonist and antagonist effects of FTY720 are exerted through up-regulation of S1P1 and translocation of S1P3. 32 Down-regulated S P1 and translocation of S1P3 from nuclear to the membrane is in parallel with increased cardiac microvascular permeability and its consequents as pathologic angiogenesis, inflammation, and pathologic conditions as sepsis, tumor growth, acute lung injury, hypoxia and microvascular complications seen in diabetes. 33,41,42 It has been suggested that fingolimod assist in the preservation of endothelial integrity of vasculature. 32 Fingolimod-induced endothelium dependent vasodilatation in the mouse aorta has been attributed to the potent activation of Akt/eNOS/NO pathway. 37 Fingolimod significantly impair flow-mediated dilation. Arterial vasodilatory function improves by discontinuation of FTY720. 33 Through beneficial interaction with PDGF and VEGF, FTY720 exhibited good anti-tumor and anti-angiogenesis properties. 39 By http://ijnl.tums.ac.ir 4 July barrier stabilization, diminishing invasion, migration and capillary tube formation in human umbilical vein endothelial cells at very low doses of FTY720, tumor metastasis has suppressed in vitro. 43 Since, S1RPs are expressed on the surface of the alveolar epithelium and lung capillary endothelium, which are involved in regulation of alveolar-capillary barriers and smoothmuscle cell tone and hypertrophy, fingolimod seems to reduced pulmonary capacity. 14 In FREEDOMS and TRANSFORMS trials administration of 0.5-1.25 mg of FTY720 has been associated with minor changes on forced expiratory volume in 1 s (FEV1) after 1 month therapy, which remained stable thereafter. Other parameters of pulmonary function test were unchanged. 31 A dose-dependent decrease in FEV1 was seen on day one with methacholine challenge test (methacholine dose of 0.25-25 mg). No bronchodilatory response to inhaled albuterol was also seen. These data suggest that fingolimod dose is ineffective on increased bronchial hyperreactivity and no paradoxical increase in airway resistance to albuterol challenge is elicited by fingolimod treatment. Dyspnea and asthma attacks were seen more frequently by 5.0 rather than 1.25 mg fingolimod. Fingolimod, larger reductions in FEV1 from baseline were seen compared with placebo (8.8% vs. 1.9%). 44 It should be noted that and chemically modified from a Chinese herb traditionally used for the treatment of asthma. 45 However, this was transient and limited to the first few weeks of administration. Overall, pulmonary effects of fingolimod are mild and with minimal clinical impact at the dose applied for treatment of MS. 45 Atheroprotective Effects of FTY720 Fingolimod has been shown to decrease atherosclerotic wall changes in apolipoprotein Edeficient mice. 32 FTY diminished atherosclerosis plaque volume and its macrophage and collagen content in mice with hypercholesterolemic diet. 46 Short-term low-dose oral FTY720 significantly reduced early development of atherosclerosis in mice. 47 In these mice, blood concentration of antiinflammatory cytokines was increased. FTY720 inhibits sphingosine kinase independent from S1PRs, which leads to the cell apoptosis via modulation of ceramide sphingosine-S1P rheostat. 48,49 Indeed, some anti-angiogenic effects of FTY720 have been reported thorough blocking S1PRs involved in recruitment of mural cells during angiogenesis. Since S1PR3 is predominantly expressed on the surface of adult rat VSMCs, FTY720 can inhibit migration of VSMCs. S1Pblokage using FTY720 weakly affects VSMCs spatial organization. Combined inhibition of PDGFR and S1PR1/3 completely abolish network forming capabilities of VSMCs, which make them good candidates for anti-angiogenic and anti-atherosclerotic treatments due to the fundamental roles of VSMC migration in these processes. 49 Indeed, FTY720 has been reported as a cardio-protective agent through reliving either tachyarrhythmia or bradyarrhythmia induced by cardiac ischemia/reperfusion injuries. 50 It improves recovery of cardiac function after myocardial ischemia-reperfusion Applied FTY720 during reperfusion decreased left ventricular end diastolic pressure and vice versa increased mortality due to induction of fatal arrhythmia without reduction of infarct size. Pretreatment with FTY720 before ischemia diminished pro-arrhythmia without any effect on infarct size. Thus, FTY720 has a potential role in preconditioning and post-conditioning. 51 The underlying mechanisms of this cardioprotection are unknown but it has been suggested that FTY720 cardioprotection pass through S1P cascade to p-21 activated kinase (Pak1), Akt cascade and inhibitory G protein Gi). 52,53 Pak1 a Ser/The kinase downstream of small G proteins is activated in a time-and dosedependent manner through sphingosine. Pak1 activity regulates cardiac channel activity and contractility, cytoskeletal dynamics, cell motility, growth and proliferation. FTY720 directly activate Pak in cardiac cells without primary conversion to FTY720 phosphate. 52 Indeed, pharmacologic dose of FTY720 (10 mg/kg/day) resists load stress-induced murine hypertrophic remodeling without deterioration of the cardiac function through Pak1 activation. Significant decrease in HW/TL ratio and in mean cross sectional area was seen by FTY720 administration.
Attenuating cardiac hypertrophy and halting transition to heart failure with preservation of cardiac function is very beneficial for prevention and treatment of cardiac hypertrophy, since according to Laplace's law limited cardiac hypertrophy might be parallel with chamber dilation and cardiac deterioration. Pak1 is a critical signaling hub in cardioprotection, which is mutually involved in limitation of excessive hypertrophic remodeling. Thus, both anti-hypertrophic and survival signals are conveyed from small GTPases to JNK pathway in cardiomyocytes by activated. The possible downstream molecule for Pak1 in this signaling path seems to be Cdc42. 54 These data hints to the application of FTY720 as a non-toxic compound with oral bioavailability in the prevention and/or treatment of cardiac disorders in high-risk patients. In addition to beneficial effects of FTY72 in cardiac disorders, FTY720 has been shown to be promising for the treatment of ischemic stroke. 55 In this case, protection of neurovascular unit in stroke, reduction of infarct lesion size and improvement of neurological function has been seen using FTY720. Diminished infiltration of immune cells and reduced apoptotic cell death in ischemic stroke lesions is in parallel with http://ijnl.tums.ac.ir 4 July improved neurologic functions using FTY720 in ischemic strokes. 56

Effects of FTY720 on Cardiac Transplantation
Outcomes FTY720 up-regulates different intracellular protective molecules as heat shock proteins which lead to stabilization of endothelial layer and decreased sensitivity of endothelial cells to inflammatory cytokines. This prevents organs from transmigration of activated inflammatory cells across endothelial layer and paranchymal infiltration, which consequently results in organ salvage. 59 Thus, FTY720 has been proposed as an immunosuppressant agent for organ transplantation purposes. Rejection, infection and drug toxicity are the leading cause of morbidity and mortality in cardiac translation cases. Indeed, the major cause of late graft failure is graft atherosclerosis. 57 In this case, a novel, less toxic, and more potent immunosuppressive agent than traditionally used cyclosporine A is developed for survival of the transplanted organ. Fingolimod decreases recirculation of lymphocytes from lymph nodes to inflammatory lesions and graft sites. 55 Fingolimod exert beneficial effects in transplant recipients beyond immune suppression. Prophylactic administration of FTY720, prolonged the survival of heart allograft transplantation if treatment begin immediately post-transplant. 31 This graft survival is distinctly dose-dependent from no effect at the lowest dose of 0.3 mg/kg/d to remarkable protection at the highest dose. Indeed, administration of oral capsule of FTY720, prolonged survival; of murine cardiac allograft, as a rescue therapy for acute rejection. 55 For acute rejection, highest dose of FTY720 is needed. Both early and late graft coronary artery diseases are attenuated using FTY720 treatment. Thus, higher longterm graft acceptance seems achievable using FTY720. Since about of patients who survived 5 years after cardiac transplantation demonstrate significant atherosclerosis on routine coronary angiography, coronary artery disease is considered as a major problem in cardiac transplantation. Graft atherosclerotic lesions are mainly due to immune-mediated damage due to previous acute rejection or persisted immunemediated reactions in chronic post-transplant phase. Perivascular cuffing and intraluminal accumulation of mononuclear leukocytes, markers of coronary vasculitis, are attenuated using FTY720. 57 This protection is achieved through dramatic reduction in PBL. 58 Another mechanism for allograft atherosclerosis is hyperproliferation of smooth muscle cells, but at clinically relevant doses of FTY720, SMC apoptosis has not been observed. 59 Fingolimod has synergistic effects with calcineurin inhibitors as cyclosporine-A (CsA) and or mammalian target of rapamycin inhibitors as sirolimus. 60 Of course, the mechanism of action of FTY720 is different from other above mentioned agents. Only mild suppression of interferon-gamma and interleukin-2 production and interference in their function is seen using FTY720 treatment. 57 Continuous simultaneous application of FTY720 and cyclosporine-A (CsA) abrogate graft atherosclerosis and chronic graft rejection. 2,57 For combination therapy, low dose of FTY720 is also effective. 54 Combined FTY720 plus CsA has been demonstrated to be well-tolerated and suppressed completely development of graft vessel disease. 61 It allows reduction in CsA dosage in patients with impaired renal/hepatic side-effects. 62 The efficacy of FTY720 is similar with mycophenolate mofetil (MMF) for prevention of graft atherosclerosis, but less bradycardia seen with FTY compared with MMF. 42,57, Maintenance mono-therapy with FTY720 modestly ameliorated chronic rejection, but was not enough for treatment of chronic rejection. 63 Less lymphocytic infiltration has been showed in cardiac allografts by administration of FTY720 for rescuing acute rejection. 57 Since it does not impair T-cell activation, proliferation and memory response to systemic viral infection it protects grafts from failure without induction of generalized immune suppression and consequent predisposition to infections. 31 FTY720 treatment was not associated by increased change in rate, nature and severity of infection. Indeed, in the case of severe decrease in leukocyte count, recovery of leukocyte count occurs within only 14 days cessation of FTY720. 64 The efficacy of FTY720 in human cardiac transplantation should be investigated. As a strong immunosuppressant agent, FTY is shown to be beneficial in the treatment of acute experimental myocarditis without induction of excessive viral replication. 59

Vascular Complications of FTY720
Regarding vascular disorders related to fingolimod, retinal arterial vasospasm and retinal vein occlusion has also been reported. 38,39 During platelet activation, S1P is released by platelets. 32 S1P exerts vasoconstrictive effects on basal arterial tone in isolated arteries through S1P2 and S1P3 receptors. These vasoconstrictive events have been attributed to increased intracellular calcium release and its consequent contractile effects on smooth muscle cells. This induces modest hypertensive effects in longterm. Cerebrovascular constriction is also seen using FTY720 treatment. Interestingly, intramedullary infusion of FTY720 has been shown to prevent hypertensive nephropathy through enhancing sodium excretion as a diuretic agent. It also decrease proteinuria and exert anti-fibrotic effects in renal tissues. 65

Effects of FTY720 on Endocrine Functions Related to its Cardiovascular Effects
FTY720 is suggested to exert potent influence on vascular homeostasis comparable to VEGF, due to the activation of a family protein kinase C (PKC). Since deregulation of S1P1/3 is responsible for cardiac microvascular complications in diabetes, cardiac functions could be improved using FTY720 in this setting via over-expression of S1P1 and enhanced translocation of S1P3. 2 FTY720 decrease the expression of PKCBII, which is involved in the pathogenesis of diabetic microangiopathy. 30,31 This effect is attenuated by expression of PKCBII on endothelial cells. FTY720 might be a beneficial agent for treatment of cardiac microvascular diseases in diabetic patients. 30 Endothelial cell dysfunction and enhanced endothelial cell permeability seen in the setting of diabetic angiopathy has been improved by application of fingolimod. 60 Indeed, reduced blood glucose level in diabetic mice has been demonstrated using FTY720 administration. FTY720 showed promising benefits in prevention of T1D related to retention of lymphocytes in the lymph nodes. However, this effect is continued prior to the occurrence of overt hyperglycemia. After disease onset, FTY720 slowed progression of disease. 60 Sustained endothelial barrier stabilization under hyperglycemic condition is seen by FTY720 application. 53 It is unknown whether FTY720 is useful in the treatment of metabolic syndrome and its consequent complications. It has not been associated with increased risk of nephrotoxicity, hepatotoxicity, pancreatic toxicity, diabetes, and myelosuppression. 59 Hyperlipidemia has not yet been reported using FTY720 treatment. Figure 1 depicts cardiovascular manifestations of FTY720 briefly. Most surveys on cardiovascular manifestations of fingolimod are performed on volunteers with healthy cardiovascular system, and there is a little guidance on probable side-effects of fingolimod in cases with pre-existing cardiovascular diseases. For cardiac risk stratification, cardiovascular monitoring include one electrocardiogram before the first dose and 6 h post-outpatient hospital cardiology visit at the first day of drug initiation (strict follow-up of blood pressure and heart rate every hour, especially at the first 6 h post-dose). 57 In patients with cardiovascular risk factors, treatment should be initiated only if benefits outweigh potential. For these cases, adequate patient monitoring is recommended after the first dose. 65 In cases with compromised cardiac functions, FTY720 is contraindicated. 57

Conclusion
Beneficial effects of fingolimod could be higher than its cardiovascular complications through administration of this agent under close observation regarding its sideeffects on cardiovascular system. Indeed, we advise clinicians to report all of the cardiovascular manifestations they encounter using FTY.

Conflict of Interests
The authors declare no conflict of interest in this study.


extfieldsofstudy: Biology Medicine

text:
Where Does Nε-Trimethyllysine for the Carnitine Biosynthesis in Mammals Come from?

Nε-trimethyllysine (TML) is a non-protein amino acid which takes part in the biosynthesis of carnitine. In mammals, the breakdown of endogenous proteins containing TML residues is recognized as starting point for the carnitine biosynthesis. Here, we document that one of the main sources of TML could be the vegetables which represent an important part of daily alimentation for most mammals. A HPLC-ESI-MS/MS method, which we previously developed for the analysis of NG-methylarginines, was utilized to quantitate TML in numerous vegetables. We report that TML, believed to be rather rare in plants as free amino acid, is, instead, ubiquitous in them and at not negligible levels. The occurrence of TML has been also confirmed in some vegetables by a HPLC method with fluorescence detection. Our results establish that TML can be introduced as free amino acid in conspicuous amounts from vegetables. The current opinion is that mammals utilize the breakdown of their endogenous proteins containing TML residues as starting point for carnitine biosynthesis. However, our finding raises the question of whether a tortuous and energy expensive route as the one of TML formation from the breakdown of endogenous proteins is really preferred when the substance is so easily available in vegetable foods. On the basis of this result, it must be taken into account that in mammals TML might be mainly introduced by diet. However, when the alimentary intake becomes insufficient, as during starvation, it might be supplied by endogenous protein breakdown.

Introduction N e -trimethyllysine (TML) is a non-protein amino acid which has an important role as metabolic intermediate being recognized as the precursor of carnitine, a metabolite essential for the fatty acid transport and utilization in mitochondria. Carnitine is biosynthesized through a sequence of four enzyme catalyzed reactions. In the first reaction, catalyzed by the N e -trimethyllysine hydroxylase, TML is converted into 3-hydroxy-TML (HTML). Then, a specific aldolase cleaves HTML into glycine and 4-Ntrimethylaminobutyraldehyde, which is successively oxidized to 4-N-trimethylaminobutyrate. Finally, the hydroxylation of the 4-Ntrimethylaminobutyrate produces 3-hydroxy-4-N-trimethylaminobutyrate, which is better known as carnitine [1]. In some animals, especially carnivores, carnitine is largely introduced with the diet and a recent study, which stimulated a considerable debate in the scientific community, suggested the possibility that the intestinal metabolism of exogenous carnitine could promote atherosclerosis in humans [2]. However, it is recognized that mammals synthesize carnitine endogenously and, to date, no biosynthetic route alternative to that reported above is known. The biosynthesis of TML has been a puzzling biochemical problem for long time. In fact, attempts aimed to demonstrate in higher organisms its formation from lysine methylation have been unsuccessful so far. Actually, the presence of an enzyme which methylates lysine by using S-adenosylmethionine as a methyl donor and forming N etrimethyllysine was reported only in the mould Neurospora crassa [3].
Since then, neither other reports in the scientific literature describing a similar enzyme in some other organism nor more information on the Neurospora crassa enzyme nor its cloning has appeared.
To date, the generally accepted view is that the metabolic source of TML is the hydrolysis of proteins which contain TML as a post-translational modification of some lysine residues. It is well known, in fact, that in mammals proteins such as calmodulin, histones, cytochrome c, and myosin contain TML residues [4,5]. Therefore, it is conceivable that free TML may be released in the course of the protein turnover and, then, utilized for carnitine production, although a direct evidence of this formation route has never been reported. Indeed, only experiments, conducted on exogenous proteins, chemically radiolabeled in vitro by the incorporation of [ 3 H]methyl groups into lysine residues and then perfused into animal livers, showed that free TML was released [6][7][8]. On the other hand, it would have been difficult to imagine another origin for TML, as this amino acid is believed to be rather rare in its free form. Actually, in the natural world, besides animals, there are few other sources known to contain free TML. As matter of fact, the occurrence at fair level of TML, as a free amino acid, has been reported in seaweeds, especially those of the Laminaria genus. For this reason, TML has also the trivial name of laminine [9,10].
Recently, we showed for the first time the occurrence of N Gmethylated derivatives of arginine in the most important vegetables utilized for human nutrition [11]. Intriguingly, also these compounds are believed to be formed in human organism by degradation of proteins containing N G -methylated arginine residues arising from post-translational modifications [12]. In the course of this study, we developed a rapid HPLC method to separate, before quantification by ESI-MS/MS, N G -methylarginines in short times without need of derivatization. During the analysis of numerous extracts of vegetables important in human nutrition, we invariably noticed in the chromatogram the presence of a peak much more retained and intense than those of arginine N G -methylated derivatives. Here, we report the identification of this peak and show that it corresponds to TML, which, therefore, appears to be ubiquitous, not at negligible levels, in all the vegetal species examined.

Materials and Methods
Reagents N e -trimethyllysine, N G -monomethylarginine, homoarginine, formic acid, ammonium formate, and the 0.1% solution of formic acid in water used for the LC-ESI-MS analyses were from Sigma-Aldrich (Milan, Italy). The standard mixture of L-amino acids, containing Ala, Arg, Asp, Glu, His, Ileu, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, Val, and Cys at 2.5 mM concentration in 0.01 M HCl, was from Pierce. AccQ FLuor reagent was from Waters (Milan, Italy). Milli-Q water was used for all the preparations of solutions and standards.

Vegetable sources
Fruits, leafy vegetables, potato (tuber), flours of Gramineae and Leguminosae were purchased in various local supermarkets. Four different lots of 1 kg of each vegetal source were analyzed at various times. Samples of alfalfa (Medicago sativa) and nettle (Urtica dioica) plants were supplied by the city botanical gardens. All vegetables used are reported in Table 1.

Sample preparations for TML determinations by LC-ESI-MS/MS
Citrus fruit juices were prepared by hand squeezing the fruits. Juices were previously centrifuged at 120006 g for 15 min and then the supernatants were diluted 1:1 with a 0.2% solution of formic acid in water. As for flours and leafy vegetables, the samples were homogenized in a mixer with formic acid 0.2% v/v in 1:3 (w/w) ratio, whereas, for fruits, the edible part (200 g) was homogenized in a mixer with formic acid 0.2% v/v in 1:1 (w/w) ratio. The homogenates were kept for 2 h under constant agitation and then centrifuged at 180006 g for 30 min at 4uC. The supernatants were finally frozen and kept at 220uC until used for analytical determinations.   by utilizing 75% of Sol. A (0.1% formic acid in water) and 25% of Sol. B (100 mM ammonium formate in water titrated to pH 4.5 with formic acid). The retention times and peak areas of the monitored fragment ions were determined by the Agilent software Chemstation version 4.2. Recovery of TML from the various matrices was determined by analyzing portions (10 g) of homogenized samples. Portions were spiked with a know amount of TML in formic acid 0.1%. The standard solutions added to the samples were 2 mmol/L. Samples of the same homogenized matrix without the addition of TML were also analyzed. The recovery of TML was obtained separately. Percentage recoveries were based on the difference between the total amount in the spiked samples versus that in the unspiked samples. Reproducibility was assesses by the determination of recovery of six individual samples. The limit of detection of TML was assessed by utilizing only the standard solutions in formic acid 0.1%. The limit of detection was determined as the concentration of TML which gave a peak height three times that of the background noise.

Determination of TML and amino acids by LC with fluorescence detection
TML quantification in the samples was also performed by reverse-phase HPLC employing a Waters instrument mod. 2690 equipped with the fluorescence detector mod. 474. The juice or extract samples of about 10 mL were centrifuged at 120006 g at 4uC for 10 min. Then, 5 mL of supernatant was filtered and loaded on a column (561 cm) filled with Bio-Rad AG 50WX8-(H + ) resin. After loading, the column was washed with five volumes of Milli-Q water and, then, the amino acids were one-step eluted with 10 mL of 15% ammonia solution in water. In order to exhaustively remove ammonia which heavily interfered with TML determination, after drying the eluate in a rotavapor, the residue was dissolved in 2 mL of NaOH 0.01 M in water and then vacuum dried again. The residue was finally dissolved in 2 mL of 0.01 M HCl in water and the solution filtered through a 0.45 mm filter. The derivatization of TML in the samples was accomplished with AccQ (6-aminoquinolyl-N-hydroxysuccinimidyl carbamate), which is usually employed in the quality control of fruit juices for the determination of the free amino acids by HPLC with fluorescence detection [13]. AccQ-N e -trimethyllysine was fluorimetrically detected by excitation at 350 nm and emission at 395 nm. AccQ-N e -trimethyllysine was identified on the basis of the retention time and quantified by comparison of the sample peak area with the calibration curve.

Determination of TML in vegetable sources
The quantification of TML in the vegetal matrices was performed by the same chromatographic procedure that was advantageously employed to analyze N G -methylated arginine derivatives in vegetables [11]. This procedure has proven to be suitable for polar substance analysis with ESI-MS/MS detection and does not require sample derivatization. The chromatography was performed with a short silica column (Supelcosil TM LC-Si), employing isocratic elution conditions. When analyzing the content of methylarginines in vegetables, multiple reaction monitoring was used for the analyte quantification. The transition utilized for N G -monomethylarginine (NMMA) MS 2 quantification was 189.2R74. Incidentally, the m/z of protonated TML is 189.2 too. In all vegetable samples, this lucky coincidence allowed the observation of an intense peak in the total ion chromatogram (TIC), emerging at a retention time (r.t.) of about 15 min, much more retained than that of NMMA. As an example, the MS 2 total ion chromatogram (TIC) by isolating at m/z 189.2 from a sample of sweet pepper berry extract is reported (Figure 1, panel A). The MS 2 fragmentation pattern of the peak at r.t. 15 min (Figure 1, panel A) shows only three fragments at m/z 130, 84, 60 ( Figure 1 panel C) which are typical of N e -trimethyllysine MS 2 fragmentation [14], as also confirmed by comparison with the fragmentation pattern of the TML standard solution. The smaller peak at r.t. 6.0 min (Figure 1, panel A) corresponds to N G -monomethylarginine. Anyway, besides the identity of the MS 2 fragmentation pattern with that of TML standard solution, the identification of the unknown peak was also confirmed by the identity of its retention time with that of an authentic TML standard solution (Figure 1, panel B). Moreover, when changing the chromatographic conditions by varying the ammonium formate concentration in the eluent, it was found that the retention time of TML on the LC-Si column strongly varied depending on the concentration of ammonium formate in the eluent, as also observed for methylarginines [11]. Therefore, in order to further confirm the peak identity, various eluent composition changes were tried. In each case, the retention times of the standard TML peak and that of the unknown compound varied in the same way. However, besides NMMA, TML is also isobaric with homoarginine (HAG), a substance also reported to occur in some vegetables such as grass pea (Lathyrus sativus) [15] and lentils [16]. In order to exclude the possibility to mistake homoarginine for TML, we run HAG standard solution in the same chromatographic conditions. We found that HAG shows a much shorter retention time (about 5.5 min) than TML, thus the two compounds are completely resolved in the chromatographic conditions we utilized (Figure 1,  panel B). This is important as the fragments at m/z 130, 84 and 60 present in MS/MS fragmentation pattern of TML also occur in the MS/MS fragmentation pattern of homoarginine, besides other fragments (Figure 1, panel E). Therefore, a complete chromatographic resolution is mandatory for a reliable attribution and quantification when both compounds are present in the same sample. Our results show that TML is present in all the vegetables analyzed (Table 1), as demonstrated for each vegetable source by the comparison of retention times and MS 2 fragmentation patterns with the authentic standard. In particular, the highest concentration was found in sweet pepper fruit. It is interesting to note that fruit juices, largely utilized in human nutrition, such those of orange and grapefruit, also contain levels of TML in the same range of the content of some essential amino acids, such as lysine and leucine [17][18][19][20][21][22]. Also, it is of interest the observation that one of the highest level of TML was found in leaves of alfalfa, a plant of paramount importance for the cattle feeding.

Evaluation of the percentage recoveries
In order to check the performance of the analytical procedure, the percentage recoveries, the reproducibility and the limits of detection for TML were assessed. Recoveries of TML, determined as reported in Materials and Methods section, were in each case higher than 95% for all analyzed samples. The reproducibility, assessed by calculating the standard deviation of six recoveries, resulted less than 5%. The detection limit, determined as that concentration of TML which gave a peak height three times that of the background noise, was 10 nmol/L.
The presence of TML in the vegetable samples was further confirmed by an independent analytical approach, based on HPLC with fluorescence detection, commonly used to analyze amino acids in food sources [13]. This procedure involves the purification of the vegetal extract by a passage on a short column of AG50WX8 (H+ form). Moreover, this procedure also allowed to have an estimation of the matrix effect in the analyses of some samples by comparing the results obtained by HPLC-ESI-MS/MS with and without the purification passage on the AG50WX8 column (see Materials and Methods). In all cases, the samples without the passage on column showed values from 2% to 15% lower than the purified samples. An important aspect of TML analysis by HPLC with fluorescence detection consists in the sample treatment after the passage on the AG50WX8 column. Generally, for amino acid analysis, the column is eluted with a 15% ammonia solution. Afterwards, the eluted solution is vacuum dried and the residue is suspended in a suitable solvent and derivatized. However, we found that in this way a broad peak, due to residual ammonia presence, heavily interfered with determination of derivatized TML, as both substances emerged in the same time range. Instead, the treatment with a diluted solution of sodium hydroxide, as described in Materials and Methods, exhaustively removed ammonia from the dried samples and completely eliminated such interference (Figure 2). Results obtained by HPLC-ESI-MS/MS and by HPLC with fluorescence detection were also compared for tomato berry, alfalfa leaves and sweet pepper berry extracts (Figure 2). The HPLC analyses confirmed the presence of TML in these vegetables at levels comparable within +/210% to those obtained by HPLC-ESI-MS/MS.

Discussion
In this paper we reported the occurrence of TML in a consistent number of vegetables and vegetable derived products. Specifically, in the samples examined, free TML levels were in the same range as other free amino acids [17][18][19][20][21][22][23][24]. This result is of noticeable interest as the presence of free TML in vegetables, excepting seaweeds, was practically ignored so far. To date, only one study reports unequivocal identification and quantification of free TML in a plant extract [23]. On the contrary, several studies report in some plants the occurrence of proteins containing the TML Occurrence of N e -Trimethyllysine in Vegetables PLOS ONE | www.plosone.org residue in their primary structure as a post-translational modification of lysine residues. Among these proteins, there are calmodulin from Papaver somniferum and Euphorbia lathyris [24,25], and cytochrome c from wheat germ, which has been found to contain two TML residues [26]. Another important protein, owing to its essential role in carbon dioxide fixation in the vegetal cell chloroplasts, is the ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCo) which, in some plant species, has been found to contain TML in the N-terminal region of its large subunit [27,28]. Since the biosynthetic pathway for TML formation, as free amino acid, is still unexplored in plants, it cannot be ruled out that the free TML also in plants origins from the breakdown of TML residue containing proteins, as it is supposed for mammals. The role of free TML in all plants we examined can be at the present only hypothesized. It may be supposed that, at least in part, TML is utilized for carnitine biosynthesis, as it happens for mammals. In fact, it has been recently demonstrated that in Arabidopsis thaliana the carnitine biosynthetic pathway shares similar features with the pathway of mammals and fungi [23]. An other possible role of TML could be that to be part of the pool of osmolytes, as it is hypothesized for seaweeds [10], and thus it could play a stress protective function in the vegetal cells. Anyway, whatever the TML role in plants may be, an important question arises, as a consequence of our results, about the true source of TML in mammals.
The current opinion is that mammals utilize the breakdown of their endogenous proteins containing TML residues as starting point for carnitine biosynthesis (Figure 3). But is it conceivable to resort to such tortuous and energy expensive route when the substance is so easily available from vegetable foods? This consideration even better applies to herbivorous mammals for which the intake of free TML through foods should be higher than for other mammals. From this point of view, it is of interest our finding that alfalfa leaves, one of the most important forage grass for the cattle feeding, resulted one of the richest source of free TML among the vegetables examined (Table 1). In this respect, besides the consideration that the amount of TML from vegetables assumed by the herbivores seems sufficient for their carnitine biosynthesis, it is also important to point out that rat and guinea pigs orally supplemented with free TML have an increased rate of carnitine biosynthesis and excretion [29][30][31]. Conversely, dietary proteins (casein, soy proteins, and wheat gluten) were found to be poor source of TML [31]. In light of such evidence, our hypothesis seems founded on a firm ground as it has already been proved that animals orally supplemented with free TML showed increased rates of carnitine biosynthesis and excretion [29,31].
On the other hand, also for humans with the particular dietary habit to eat only vegetable foods (vegetarians or vegans), it is hardly conceivable that only endogenous TML can suit the needs for their carnitine biosynthesis. Indeed, it has been shown that vegetarian adults have slightly reduced plasma carnitine levels compared to omnivorous individuals [32,33]. However, serum carnitine levels are more markedly depressed in vegetarian children [32] and in premature infants who do not receive a dietary source of carnitine [34]. However, this could be ascribed to immaturity of the biosynthetic conversion machinery of TML into carnitine. Lastly, a warning that could be relevant to vegans is that the assumption of free TML through vegetables consumption may represent a risk factor for those subjects affected by dysregulation of the carnitine biosynthesis due to a mutation of the N etrimethyllysine hydroxylase gene [35].
In conclusion, although it cannot be excluded that a certain amount of TML might be produced by the hydrolysis of endogenous proteins containing such residue as a posttranslational modification, it does not appear likely that this route might be the preferred one when the substance is so easily available through a vegetable containing diet ( Figure 3). Conversely, when the dietary intake become insufficient, it cannot be ruled out that the breakdown of endogenous proteins could become the prime source of TML. From this point of view, it seems reasonable to suggest that in mammals TML utilized for carnitine biosynthesis might be mainly taken from diet (for herbivore animals this should be even more likely). However, when the alimentary intake fails, as in case of starvation, TML might be produced from endogenous protein breakdown, similarly to the essential amino acids when their intake becomes insufficient. The biochemical pathway, according to the view generally accepted, entails, as the metabolic source of TML in mammals, the hydrolysis of proteins which contain TML as a post-translational modification of some lysine residues. Right side: The vegetal food intake, as suggested in this paper, directly provides mammals with the TML to be converted into carnitine. Circles depict amino acids. The black filled circle indicates lysine. AdoMet: S-adenosylmethionine. Both protein and AdoMet syntheses require ATP (indicated in parentheses). doi:10.1371/journal.pone.0084589.g003