European Journal of Pharmacology 730 (2014) 26–30

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Endocrine pharmacology

Elevated melatonin levels in natalizumab-treated female patients with relapsing-remitting multiple sclerosis: Relationship to oxidative stress Carmen Bahamonde a,1, Cristina Conde a,1, Eduardo Agüera a, Rafael Lillo b, Evelio Luque c, Félix Gascón d, Montserrat Feijóo e, Antonio H. Cruz d, Fernando Sánchez-López a, Isaac Túnez e,n a Department of Neurology, Maimonides Institute for Research in Biomedicine of Cordoba (IMIBIC)/Reina Sofia University Hospital/University of Cordoba, Spain b Department of Sociosanitary, Radiology and Physical Medicine, Psychiatry Section, Faculty of Medicine, University of Cordoba, Spain c Department of Morphological Sciences, Histology Section, Faculty of Medicine/ IMIBIC/Reina Sofia University Hospital/University of Cordoba, Spain d Department of Clinical Analysis, IMIBIC/Regional Hospital of Pedroches Valley/University of Cordoba, Spain e Department of Biochemistry and Molecular Biology, Faculty of Medicine/IMIBIC/Reina Sofia University Hospital/University of Cordoba, Spain

art ic l e i nf o

a b s t r a c t

Article history: Received 22 October 2013 Received in revised form 14 February 2014 Accepted 17 February 2014 Available online 25 February 2014

Natalizumab is currently the most successful clinical treatment for multiple sclerosis. The use of this drug is associated with the reduction in the number of relapses and a slowing in disease progression, as well as an improvement in signs and symptoms displayed by the patients. To evaluate the effect of natalizumab on melatonin and its relationship with peripheral oxidative damage, we studied the serum melatonin levels in 18 patients with relapsing-remitting multiple sclerosis. Natalizumab caused significant increases in serum melatonin concentrations. This change was associated with a rise in increase of antioxidants and a reduction in oxidative stress biomarkers. In conclusion, these data may explain, at least in part, some of the beneficial effects exhibited by disease antibody such as its antioxidant capacity. & 2014 Elsevier B.V. All rights reserved.

Keywords: Melatonin Natalizumab Oxidative stress Relapsing-remitting multiple sclerosis Tysabris

1. Introduction The most common cause of non-traumatic neurological disorder in young adults is multiple sclerosis. Its pathogenesis involves inflammation, oxidative stress, demyelinization and neuronal loss (Compston and Coles, 2002, 2008; Miller, 2012). There are four types of MS: (i) progressive relapsing, (ii) primary progressive, (iii) relapsing-remitting, and (iv) secondary progressive (Compston and Coles, 2002, 2008). Oxidative stress seems to play an important role in this neurodegenerative condition. Thus, reactive oxygen species have a relevant function in multiple sclerosis pathogenesis (Melamud et al., 2012; Miller et al., 2012; Tasset et al., 2012a, 2012b). Our recent studies found that multiple sclerosis patients have elevated levels of oxidative stress biomarkers, together with a global antioxidant deficiency. This supports the hypothesis that an imbalance between reactive oxygen species and antioxidant

n Correspondence to: Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Córdoba, Avda. Menéndez Pidal s/n, 14004 Córdoba, Spain. Tel.: þ 34 957 21 82 68; fax: þ34 957 21 82 29. E-mail address: fm2tufi[email protected] (I. Túnez). 1 Equal contributor.

http://dx.doi.org/10.1016/j.ejphar.2014.02.020 0014-2999 & 2014 Elsevier B.V. All rights reserved.

system precedes the inflammatory response, at least, in terms of a relapse (Tasset et al., 2012a, 2012b). Environmental factors have important role in multiple sclerosis pathogenesis (Ghorbani et al., 2013; Hedstrom et al., 2011). Studies have shown a reduction in blood melatonin (N-acetyl-5-methoxytryptamine) levels and dysregulation of its synthesis, secretion and circadian rhythm associated with fatigue, depression and sleep disorder in multiple sclerosis patients (Akpinar et al., 2008; Compston and Coles, 2002; Ghorbani et al., 2013; Melamud et al., 2012; Sandyk and Awerbuch, 1993). Melatonin produced by pineal gland and elsewhere is a potent free radical scavenger and antioxidant (Fischer et al., 2013; Galano et al., 2011, 2013; Miller et al., 2012) and, as such, it may be protective against the neural damage associated with multiple sclerosis. Natarajan et al. (2012) reported that the melatonin pathway genes are involved in progression of multiple sclerosis. In addition, a recent study found that its administration reduced oxidative status associated to multiple sclerosis. This was accompanied by a reported improvement in functional characteristics of patients, evaluated by the Expanded Disability Status Scale, though the difference could not be statistically verified (Natarajan et al., 2012). These data are consistent with the idea that this, melatonin, may play an important role in the development and progression of multiple sclerosis.

C. Bahamonde et al. / European Journal of Pharmacology 730 (2014) 26–30

To date, there is no cure for multiple sclerosis, so treatments are aimed at preventing relapses and mitigating the signs and symptoms associated with this disease. A variety of drugs are used in therapy; natalizumab (Tysabris) is reported to be the most effective (Cadavid et al., 2013; Phillips et al., 2011; Stephenson et al., 2012; Wickstrom et al., 2013), whereas the most common is interferon-beta. With this background, we addressed the idea that the changes produced after treatment with natalizumab may be due, in part, to the stabilization of melatonin levels and its antioxidant effects.

2. Materials and methods Eighteen patients (5 men and 13 women) with relapsingremitting multiple sclerosis were recruited for the study from the Department of Neurology at Queen Sofia University Hospital in Cordoba. The revised McDonald (Polman et al., 2005) criteria were used and the patients were treated with 300 mg natalizumab (anti-VLA-4; Tysabri, Biogen Idec, Cambridge, MA, USA) administered via intravenous infusion every 4 weeks (28 days) in concordance with current Spanish guidelines during 56 weeks (MS-56) (Fig. 1). The infusions were given between 16:00 and 18:00 hours. Clinical examination was performed using the Expanded Disability Status Scale (EDSS) (Kurtzke, 1983). Peripheral blood samples were taken immediately prior to the first infusion (baseline) and before the fourteenth infusion. Blood was collected between 15:30 and 17:30 hours in chilled BD Vacutainers tubes (Becton-Dickinson and Company, BD, Franklin Lakes, NJ, USA) without anticoagulant (for serum) or with anticoagulant, EDTA-K2 (for plasma and erythrocytes). Thereafter serum or plasma samples were immediately separated by centrifugation at 1500 g at 4 1C for 15 min and the fraction was frozen in aliquots and stored at  85 1C. 2.1. Biochemical parameters Serum samples were tested for melatonin using enzyme immunoassay (Melatonin ELISA) kits according to the manufacturer's instructions (GenWay Biotech Inc., San Diego, CA, USA). The quantity of the oxidative DNA adduct 8-hydroxy-2'deoxyguanosine (8-OHdG) was evaluated using the assay kit (8-OHdG Check-437-0122) purchased from JaICA (Japan Institute for the Control of Aging, Fukuroi city Shizuoka, Japan). Reduced glutathione (GSH) levels were evaluated using the Bioxytech GSH400 kit (Oxis International, Portland, OR, USA). The GSH concentration is based on a reaction which leads to the formation of a chromophore with absorbance at 400 nm. The total antioxidant capacity (PAO, KPA-050) was evaluated using a kit purchased from JaICA (Japan Institute for the Control of Aging, Fukuroi City Shizuoka, Japan); this assay is based on the reduction of Cu2 þ to Cu þ by the combined action of all of the antioxidants present in the sample. Thus, the chromogenic reagent forms a complex with Cu þ which has an absorbance at 490 nm. For quantitative detection of the soluble vascular cell adhesion molecule-1 (sVCAM-1) an assay kit (Milliplexs MAP Kit, Human 1st infusion

14th infusion 56 weeks 2nd 3rd 4th 5th 6th 7th 8th 9th 10th 11th 12th 13th

1st extraction (baseline)

2nd extraction

Just before 1st infusion

Just before 14th infusion

Fig. 1. Schematic of natalizumab administration.

27

Table 1 Characteristics of patients with relapsing-remitting multiple sclerosis (RRMS). Values are expressed as mean 7 S.D. Baseline: patients with multiple sclerosis previous treatment (baseline) and after 56 weeks of treatment with natalizumab (MS-56). RRMS (18) Gender (men/women) Age (years) Mean EDSS Baseline MS-56 Δ (MS-56 – Baseline) Disease duration (years) No. of relapses before treatment No. of relapses during treatment Melatonin by age (pg/ml) Baseline r 40 years 4 40 years MS-56 r 40 years 4 40 years a

5/13 40.5 7 7.8 (28–57) 4.2 7 1.5 4.4 7 1.4 0.2 7 0.8 7.6 7 3.9 (3–13) 4.6 7 1.4 0.6 7 0.8

4.4 7 1.8 7.17 2.2 8.17 3.7 17.3 7 2.2a

Po 0.05 vs baseline group.

CVVD Panel 1 96-Well Plate Assay, Cat. # HCVD1-67AK) purchased from Millipore™ (Millipore Corporation, Concord Road, Billerica, MA, USA) was used.

2.2. Statistical analysis Statistical evaluation was performed using SPSS 17.0s software (SPSS Iberica, Madrid, Spain) for Windows. Intergroup significance was determined by Wilconxon-matched pairs test to analyze nonparametric data. P o0.05 was considered significant.

3. Results The demographic features of the study groups are presented in Table 1. Our data did not find significant change between EDSS score before and after treatment. However, the EDSS increase suffered by patients after treatment presented a correlation with melatonin levels after treatment (Spearmen Rho, r: 0.777; P¼ 0.04). Mean serum melatonin pre-treatment levels in the patient group were significantly lower than after the 56 weeks of treatment: 5.0 pg/ ml in baseline vs 10.1 pg/ml after 56 weeks of treatment (Fig. 2). During treatment with natalizumab, women's levels of melatonin in serum increased significantly, 4.3 pg/ml (baseline) vs 10.8 pg/ml (after 56 weeks of treatment). Our data revealed that levels of melatonin increased by 21% in men after natalizumab treatment (Fig. 1). However, the increase caused by nataluzimab was much more intense in women, establishing much higher levels in women than in men (Fig. 2). When the patients were separated by age into two groups, we only detected significant differences between baseline and after 56 weeks of treatment situation in patients over 40 years (Table 1). In addition, our results show that serum melatonin elevation is associated with a reduction in oxidative stress markers characterized by an increase in GSH levels and a reduction in 8OHdG levels, whereas PAO did not change (Table 2). Finally, our analysis found that natalizumab induced a significant reduction in sVCAM (Table 2), as well as these data present a significant correlation between melatonin and sVCAM levels after treatment (Spearman Rho: r  0.479, P o0.05).

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C. Bahamonde et al. / European Journal of Pharmacology 730 (2014) 26–30

18

** •

16

** 14 12 10 8 6 4 2 0 Total Baseline

Total MS-56

Men Baseline

Men MS-56

Women Baseline

Women MS-56

Fig. 2. Serum levels of melatonin in patient with relapsing-remitting multiple sclerosis (RRMS). Values are expressed as mean7 S.D. Baseline: Patients with RRMS previous treatment, and MS-56: after 56 weeks of treatment with natalizumab. nnP o0.01 vs baseline group; P o0.05 vs Men MS-56 group.

Table 2 Effect of natalizumab on oxidative stress biomarkers (8-hydroxy-20 deoxyguanosine, 8-OHdG; reduced glutathione, GSH; total antioxidant capacity, PAO; soluble vascular cell adhesion molecule-1, sVCAM-1). Values are expressed as mean7 S.D. Baseline: patients with MS previous treatment, and MS-56: after 56 weeks of treatment with natalizumab.

Baseline MS-56 a b c

8OHdG (ng/ml)

GSH (μM/g Hb)

PAO (mM)

sVCAM-1 (ng/ml)

80.8 7 35.7 58.5 7 27.7b

68.9 7 24.9 86.4 7 25.1c

0.22 7 0.1 0.25 7 0.1

586.0 7188.1 86.3 727.0a

P o0.001 vs baseline. Po 0.01 vs baseline. Po 0.05 vs baseline.

4. Dicussion So far as we can determine, this is the first evidence which shows that natalizumab affects circulating melatonin levels. Our findings reveal that natalizumab caused an increase in melatonin concentration, which was associated with an oxidative stress and adhesion molecule reduction. In addition, we observed the maintenance of EDSS during treatment with natalizumab, a situation that can be interpreted with a blockade, or at least slowing, in the progression of the disease. Inadequate nocturnal levels of melatonin are associated with altered sleep–wake rhythm, aging and oxidative damage (Cardinali et al., 2012). A reduction in melatonin levels has been reported with the development of RRMS. Thus, administration may cause an improvement in disease course and associated disorders as fatigue or sleep cycle disturbances. Moreover, due to its antioxidant capacity and different physiological actions, drop in melatonin levels maybe involved with aging and neurodegenerative disease, and thus its reduction may be a biomarker of some conditions (Lin et al., 2013; Melamud et al., 2012; Miller et al., 2013; Reiter et al., 2000; Wang, 2009; Wang et al., 2013). Additionally, melatonin is characterized by presented a neuroprotective effect by mean different routes such as antioxidant, antiinflammatory, antiapoptotic and anti-excitotoxicity activities (Esposito and Cuzzocrea, 2010; Luchetti et al., 2010; Reiter et al., 2000, 1999; Rosales-Corral et al., 2012; Sewerynek et al., 1995; Wang et al., 2013). In our work, natalizumab caused a reduction in the oxidative stress biomarkers and number of relapses suffered by patients. This is consistent with other studies in which this drug prevents

new outbreaks, and more recent findings show that natalizumab administration is associated with disease improvement (Cadavid et al., 2013; Stephenson et al., 2012; Wickstrom et al., 2013). Additionally, data previously reported by our group showed that this agent induces a reduction in oxidative damage and an increase in translocation of nuclear factor (erythroid-derived 2)like 2 factor (Nrf2) into the cell nucleus (Tasset et al., 2013). Nrf2 is responsible for regulating the expression of genes encoding antioxidant protein system phase II such as heme-oxygenase 1 (HO-1), glutathione S-transferase (GST), NAD(P)H quinine oxidoreductase (NQO1) and other. The effect of natalizumab on Nrf2 has not been explained. However, the increase in circulating levels of melatonin may explain this change, considering that the pineal indole prompts Nrf2 translocation into de nucleus. Obviously, increasing melatonin levels induced by natalizumab justify and support a phenomenon previously observed by our group: natalizumab induces Nrf2 and acts as antioxidant (Tasset et al., 2012b, 2013). Melatonin has the capacity to cause translocation of this factor. In addition, these results are indirectly agreement with studies of Miller et al. (Miller, 2012; Miller et al., 2013, 2012) who found that melatonin administration reduced oxidative stress in the erythrocytes of patients with multiple sclerosis, increasing superoxide dismutasa (SOD) and glutathione peroxidasa (GPx) levels and decreasing of malonyldialdehyde (MDA) levels (Miller, 2012; Miller et al., 2013, 2012). Undoubtedly, the discovery of the effect of natalizumab on melatonin may give a better understanding of natalizumab action mechanism (Fig. 3) and is indicative that at least part of its benefits maybe partly due to its ability to act on melatonin levels (Fig. 3). While it is important to clarify the pathways and mechanisms by which natalizumab prompts changes in serum melatonin level. Both melatonin and natalizumab have been associated to an effect on adhesion factors such VCAM and intercellular adhesion molecule 1 (ICAM-1) (Constantinescu et al., 1997; Kang et al., 2001; Lin et al., 2013; Wang, 2009). Thus, administration of melatonin in animal models showed a reduction in the levels of ICAM in experimental autoimmune encephalomyelitis (Constantinescu et al., 1997; Kang et al., 2001). While most recent data show that melatonin triggers a reduction in the levels of ICAM-1and VCAM-1 in cultured endothelial aorta, a melatonin deficit due to pinealectomized is associated with increases of levels of VCAM-1, ICAM-1, matrix metallopoteinase-9 (MMP-9), monocyte chemotactic protein-1 (MCP-1) and oxidative stress biomarkers (Lin et al., 2013; Wang, 2009). Again, the data indicate that some of the effects may be triggered by natalizumab are

C. Bahamonde et al. / European Journal of Pharmacology 730 (2014) 26–30

Melatonin

29

IFNγ TNFα

Blood

CNS

Natalizumab

Neuron

VLA-4 VCAM

CD4

CD4

Leukocytes

Transmigration

Axonaldamage Macrophage

BBB

Complement

Antibody

Oxidative stress

Inflammation Mitochondria NAD(P)H-oxidase

Melatonin Melatonin:

Myeloperoxidase Xanthine oxidase

VCAM

UncoupledNOS

TNFα

Other

NFκB

NFκB ROS GSH/GSSG

Bcl-2 Bax GPx GRd

Susceptibility to activate

ICAM

GST

VCAM

NQO-1

TNFα Cell adhesion

HO-1 Nrf2-ARE pathway

Interleukines Fig. 3. This figure shows the possible biochemical and molecular mechanisms involved in multiple sclerosis and its relationship with melatonin level (A), presenting the blocking effect of natalizumab between VLA-4 and VCAM with preventing cell transmigration and exerts its protective effect. Thus, inhibition of leukocyte transmigration causes a neuroinflammation and, possibly assists and facilitates the recovery of the synthesis and release of melatonin, phenomenon which could help the patient's recuperation. This implicates that the effect of natalizumab on peripheral levels of melatonin, at least in part, might be finally causing beneficial effects observed after treatment with natalizumab, bearing in mind the different mechanisms of action of pineal indole (B). Although, the mechanisms by which this antibody would trigger this effect are not clearly known. Finally, we propose the activation of leukocytes, for subsequent migration to the central nervous system (CNS), requires a state of awareness and readiness prior derived from redox imbalance, especially glutathione, and an over production of reactive oxygen species (C). Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2; ICAM: intercellular adhesion molecule; GPx: glutathione peroxidasa; GRd: glutathione reductase; GST: glutathione S-tranferase; HO-1: heme-oxygenase 1; NFκB: nuclear factor kappa-light-cjain-enhancer of activated B cells; NO: nitric oxide; NQO1: NAD(P)H quinine oxidoreductase; Nrf2-ARE: nuclear factor (erythroid-derived 2)-like 2 factor-antioxidant response element; Od2  : anion superoxide; TNFα: tumor necrosis factor alpha; VCAM: vascular cell adhesion molecule.

mediated by changes in melatonin levels. All these findings are line with the results reported by us where melatonin levels after treatment correlate inversely with sVCAM levels. These data suggest two interesting and unique facts including as the increase of melatonin after treatment with natalizumab, and therefore from this perspective the action of natalizumab is greater in patients aged over 40 years and female. This work has limitations since a time course study with melatonin was not performed and night-time melatonin levels were not measured. However, we should mention that the blood samples were taken over a period of hours when melatonin levels are lower, so we can deduce and understand that the increase is

more relevant. In addition, another potential limitation of the small number is patients recruited. This is because treatment with natalizumab is very restrictive, which caused a reduction in the number of patients included in the study. Finally, another possible limitation is failing to evaluate the association between melatonin and its effect on the quality of sleep. Taken together, the findings indicate of the need for further studies for the purpose of improving an understanding of the mechanisms involved in the protective effects triggered by natalizumab. In brief, natalizumab-treated patients had improvement on their general condition characterized by a reduction in the number of relapses and recovery associated with major levels of melatonin

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