Elena Alvira
email: malvira@ull.es

CAPACITY OF SMALL MOLECULES TO FORM β-CYCLODEXTRIN INCLUSION COMPLEXES

Keywords: inclusion complex, potential energy surfaces, computer simulation, molecular mechanics, molecular dynamics inclusion complex, potential energy surfaces, computer simulation, molecular mechanics, molecular dynamics

published or not. The documents may come

Introduction

In previous work we proposed an analytical model for the interaction energy between α-, βand γ-cyclodextrin with guest molecules of different structure and size [1]. The intermolecular energy was then modelled by a Lennard-Jones potential which represents the attractive and repulsive interactions between all atoms and molecules in the system, and a continuum description of the cavity walls [2,3]. The difference in molecular size was represented in that study by the potential energy parameter σ , but without including any parameter related to the structure of the guest molecules. This simple model is able to reproduce several quantitative and qualitative features of the interaction energy between cyclodextrin (CD) and atoms, and cyclic or spherical guest molecules. The adsorption energy obtained from this model is only valid when the guest centre of mass is located along the cavity axis, because for points away from it there is no analytical solution for the energy. Therefore this analytical model is inadequate to analyse the mobility of these types of molecules around β-CD and even inside the cavity, because the guest does not always move along the axis.

Our aim in the present study is therefore firstly to calculate the interaction energy between β-CD and the guest molecule inside and outside the cavity, not only along the axis, using a simple pairwise-additive Lennard-Jones potential and a continuum description of the cavity walls. The Lennard-Jones potential represents the attractive and repulsive interactions which act between all atoms and molecules, even totally neutral ones; in contrast to the other types of forces that may or not be present depending on the properties of the molecules, such as electrostatic charges, dipoles or hydrogen bonds. Since continuity is assumed for the guest and the cavity, the interaction energy presented in this work is not capable of reproducing the hydrogen bonds formed by the guest molecule with CD. However, the van der Waals term is the main contribution to the total energy, particularly inside the cavity and so directly, determines the configuration of the inclusion complex. To take into account the effects of the solvent, other interaction energies must be applied.

We also examine the influence of temperature on inclusion complex formation by means of molecular dynamics (MD), since this simulation method is based on the resolution of classical equations of motion to determine the trajectories of the particles depending on the initial conditions [START_REF] Frenkel | Understanding Molecular Simulation[END_REF][START_REF] Rapaport | The Art of Molecular Dynamics Simulation[END_REF][START_REF] Allen | Computer Simulation of liquids[END_REF]. By treating the results generated in this process with statistical methods, one can obtain more detailed information about the potential parameters related to the size and composition of the guest molecule that influence formation of inclusion complexes with β-CD. In section 2 we present the theoretical methods and in section 3, their main results.

The Model

Expression of the interaction potential

The intermolecular energy is modelled by a Lennard-Jones potential, which represents the attractive and repulsive interactions between all atoms and molecules in the system, even totally neutral ones. The essential effects of the confinement should be preserved if we make the assumption of a continuum description of the cavity walls [1][2][3], therefore the interaction energy W can be calculated as: Ǻ for β-CD) [START_REF] Szejtli | Comprehensive Supramolecular Chemistry[END_REF].

( ) ( ) , mol CD mol S W r V r r dr ρ = ∫ r r r r (1)
Here the Z axis is collinear with the cone axis (thus the XY plane is parallel to the cone base), and the origin of the coordinates lies at the centre of the cavity. When we consider the interaction energy W by the integration in Eq. ( 1), we are substituting the discrete atoms of the CD by a uniform distribution of mass on the cavity surface.

Therefore, we can also replace the potential parameters for the different atoms belonging to the molecule ( ) about 2 Ǻ and the potential surface for each region is determined as the minimum energy for every point on the plane in the corresponding interval of Z.

Simulation method

Molecular dynamics (MD) is based on the resolution of classical equations of motion to determine the trajectories of the particles, depending on the initial conditions.

The initial conditions of the guest molecule are in this case determined by the centre of mass position and velocity. The magnitude of the initial velocity depends on the temperature of the process, but its direction in each trajectory and the initial centre of mass position are determined randomly. The simulation time for each trajectory is 1 ns with a step of 1 fs and the configuration and energies (kinetic and potential) were written every 100 steps. We use an in-house program written in Fortran and the equations of motion to perform constant temperature molecular dynamics are integrated numerically using a variant of the leap-frog scheme (proposed by Brown and Clarke) [START_REF] Brown | [END_REF], constraining the rotational and translational kinetic energies separately [9]. To determine the preferential binding site of the guest molecule, the number densities of presence in a volume element are calculated. We define a grid in which the distance between two consecutive points is 0.5 Å and the number of guest positions in each volume element is the resulting number density for each trajectory and for the guest [10][11][12]. The position probability density is calculated by dividing the number density in a volume element by the total number of centre of mass positions of the guest.

We calculate different trajectories with initial configurations of the guest on the exterior of the β-CD: near the primary (narrow end) and secondary rims (wide end) of the CD, and outside the cavity walls. However the molecule can enter the cavity and then form an inclusion complex only for certain initial positions of its centre of mass, and these positions are always near the rims of the CD and never outside the cavity walls. To calculate the probability of forming inclusion complex inc P , we determine the external configurations on the grid from which the molecule can enter the cavity. The rate of these positions with respect to the total number of points on the grid is the corresponding probability.

The simulation is carried out for molecules with different size and composition, and at different temperatures.

Results and discussion

Interaction energy

The interaction energy W depends on the size and composition of the guest molecule through the potential parameters (σ , ε ), and the position of its centre of mass

σ = Å,
therefore the confinement in β-CD involves a more intense interaction, greater even than for linear molecules [2,3]. For instance, if we apply the present model to cyclopentadiene, taking into account the results obtained by a force field method [13], it can be represented by the parameters 3.5 σ = Å and 0.0004 ε = eV, ε being 5 times less than for linear molecules such as acrylonitrile or methyl vinyl ketone. In Table 1 some samples of atoms and molecules are included along with some parameters that simulate the interaction between β-CD with these guest molecules [13,[START_REF] Lipkowitz | [END_REF]16].

However, the interaction energies presented in this eV, since ε only acts as a scaling factor on the potential energy W, these bindings energies are about 10 times bigger than the values corresponding to real cyclic or spherical molecules.

Figure 2 shows the potential energy surfaces for atoms, cyclic or spherical molecules with σ = 2.8 Ǻ, 0.006 ε = eV (Fig. 2a) and for σ = 3.7 Ǻ, 0.006 ε = eV (Fig. 2b). They are similar in the size of the regions where the energy is attractive, this feature being a consequence of the cavity geometry. The main differences in the potential energy surfaces are related to the magnitude of the energy, which is largely dependent on the molecular composition rather than its geometry, as occurs in the continuum model for the interaction between a linear molecule and β-CD. obtained by the all-atoms model, the main discrepancies derive from the position of the guest centre of mass in the configuration of the inclusion complex, because this continuum model is only able to reproduce the position in the plane Z = constant by the distance from the cavity axis instead of the (X, Y) coordinates.

Molecular Dynamics

The evolution of the guest molecule in each trajectory and the resultant average energy are different because the initial conditions determine the integration of the equations of motion. The initial velocity does not influence the number densities and the mean energy of the process, but the starting centre of mass position determines the behaviour of the guest in each process and decisively affects the probability of forming inclusion complex. When the guest initial position is located outside the cavity walls the molecule always stays on the outside of the β-CD where it does not reach a stable configuration, moving around the cavity continually or even tending to move away.

During trajectories whose starting molecular configuration is near the rims of the β-CD the guest tends to enter the cavity, remain inside forming an inclusion complex or exit from it after a period, depending on the potential parameters. However, inside the cavity the molecule does not reach a stable configuration because it is fluctuating around the most probable position p r r with an amplitude that also depends on parameters (σ , ε ).

This effect can be appreciated in Figure 3 are presented in Table 2.

It can be seen that the most probable configuration ranges from near the cavity walls to the centre of the β-CD as the molecular size increases: so taking into account the method used to calculate the position probability density, we conclude that it corresponds to the minimum energy configuration of the complex in this type of molecule. The mean energy for the simulation mean W of different molecules is also presented in Table 2. mean W is greater than min W because the energy of every position with probability different to zero contributes to the average energy, for bigger molecules there are greater energy differences inside the β-CD and so greater differences from min W (Fig. 4). In order to analyse the dependence of the results on the length of the simulation, Figure 4 shows the variations of the potential energy during a trajectory for different guest molecules. There are no great differences between the mean values for 1 and 2 ns (about 4 parts in 1000), however the computer time needed for carrying out the simulation is considerably shorter. If we compare the position probability density for these simulation times, we conclude that the most probable positions are the same and the distribution is similar for each case. But for 2 ns we obtain greater values for the position probability, implying therefore more stabilization of the system and similar results to 1 ns. We can conclude that a simulation time of 1ns is adequate for this system.

For molecules with 3.0 σ ≥ Å, the parameter ε only acts as a scaling factor in mean W and does not influence the position probability density. For molecules with 3.0 σ < Å, ε affects the amplitude of oscillations around the most probable position: a greater value of the parameter implies smaller fluctuations (Fig. 5). The meaning of ε The probability of forming inclusion complex inc P is presented in Table 2. The differences in inc P are due mainly to the distance from the rims of the β-CD at which each molecule must be located initially to enter the cavity, although in some cases the guest exits from the host after the inclusion time. This distance is usually further from the wide end of the β-CD, and ranges from 3 Å for 2.4

σ = Å to 6 Å for 4.1 σ = Å.
Therefore, considering the same grid around the cavity in all cases, we can conclude that larger molecules have greater probabilities of forming inclusion complexes.

Conclusions

We have studied the mobility of atoms, cyclic or spherical guest molecules In the dynamical study of the system we can conclude that the molecule can enter the cavity and then form an inclusion complex only for certain initial positions of its centre of mass, and these positions are always near the rims of the CD and never outside the cavity walls. The probability of forming an inclusion complex increases with the size of the guest molecule and the temperature of the process. The most probable configuration of the complex formed with cyclic or spherical molecules corresponds to that of minimum energy. The temperature of the simulation affects the inclusion time spent by the guest inside the cavity, when its interaction with β-CD is represented by parameters

  molecule-CD interaction is represented by an average over the uniformly distributed atoms in the CD. CD ρ is the superficial density of atoms in the CD cavity, mol r r is the position of the centre of mass for the guest molecule and r d r is the differential element of the surface (on the cavity) located at r r . The CD is considered as a truncated cone, h being the axial length ( 7 ≈ h Ǻ), b the radius of the larger base of the cone ( 5 ≈ b Ǻ for β-CD) and a the radius of the smaller top ( 4 ≈ a

  values for the composition of both systems. In order to bring out more clearly the dependence of W on the composition of the guest molecule, we do not consider the values of these parameters for β-CD and the guest separately, but represent the Lennard-Jones parameters for the interaction between the atoms of the CD and the molecule by σ and ε . As we are dealing with a conical continuum geometry, for every plane Z = constant, the potential energy is the same for points ( ) reason we characterize the guest centre of mass position by the distance d from the cavity axis and the Z coordinate. The potential energy is determined by Eq. (1) for different positions of the guest centre of mass mol r r , inside and outside the CD. In each plane Z = constant, about 500 points are explored and the range of variation along the Z axis is about 10 Ǻ. The results obtained are represented by the potential energy surfaces, penetration potentials and the , as in previous works [1-3]. The curve joining the minimum potential energy for every plane Z = constant defines the penetration potential, which describes the variation in interaction energy when its path through the cavity is non-axial. The position of the guest molecule for which we obtain the absolute minimum potential energy gives the geometry of the inclusion complex. The potential energy surfaces are represented by partitioning the variation range of the Z axis in the β-CD cavity into four parts, depending on the position of the guest molecule centre of mass near the top of the cone (region I), near the centre of the cavity (regions II and III) and near the cone base (region IV). The length of each domain is

  order to perform a comparative study we consider different values for the parameter σ to represent the interaction between β-CD and the guest, because ε only acts as a scaling factor on the potential energy W.

Figure 1

 1 Figure1exhibits the penetration potential (solid curves) for guest molecules

  potential presented in this study with the results

  , which shows the position probability for molecules with different σ and the same ε , and includes a schematic representation of the projections of the cavity in the plane . The range of oscillations inside the β-CD increases as the molecular size decreases, which means greater delocalization for the smaller values for the position probability. The maximum probability max P and the corresponding position of the guest

  the interaction is also reflected in the dynamic behaviour of the system, the molecule only spends a period (inclusion time t) inside, after which it exits from the CD and moves away. This t depends on the initial conditions and decreases as temperature increases (the mean inclusion time is 394 ps at 293 K, 381 ps at 323 K and 356 ps at 363 K).

  inc P is independent of parameter ε but the temperature T of the process influences inc P decisively, because the magnitude of the initial velocities depends on T and determines the maximum distance from the rims needed by the molecule to enter the cavity. These values for 323 T = K are approximately double those for 293 T = K, this means that inc P increases as T does although the ratio between the probabilities of different-sized molecules remains similar. The suitability of the model presented in this work can be confirmed by comparing results with those of Ref. 10 and 11, where Lipkowitz et al. studied where and how different analytes bind to permethylated β-CD in gas chromatography using different computational methods (standard molecular dynamics and Monte Carlo simulations). The structure of some considered selectands is essentially cyclic and in the , the intermolecular forces responsible for holding the host-guest complex are exclusively van der Waals forces[17]. They conclude that the preferred binding site for small analytes is the interior of the macrocycle with rapid shuttling between the primary and secondary rims of the host cavity, in accordance with the results obtained by the model presented in this study.

  inside and around β-CD by molecular dynamics simulation at constant temperature. The intermolecular energy is modelled by a Lennard-Jones potential and a continuum description of the cavity walls. It is found that the variation of the potential energy along the Z coordinate (parallel to the cavity axis), resembles a well potential where the minimum energy is lower as the atomic size of the molecule increases. The distance between the cavity walls and the guest molecule in the absolute minimum energy configuration is approximately σ 12 . 1 and ε governs the strength of the interaction.

Fig. 1 :Fig. 2 :

 12 Fig.1: The penetration potential (solid curves) and the interaction energy along the cavity axis (dashed curves) for guest molecules with different σ and

Fig. 3 :

 3 Fig. 3: Projections on the XY and XZ plane of the probability density of presence for guest molecules with different σ ,

Fig. 4 :Fig. 5 :

 45 Fig.4: The potential energy during a trajectory for guest molecules with different σ

Table are

 are

	obtained with	ε =	0.006

Table 1 .

 1 The minimum energy min W , the centre of mass position in the minimum energy

	σ <	3.0	Å and	ε ≤	0.003	eV.

Table 2 .

 2 The maximum probability max P , the most probable configuration of the guest

	p r r	=	(	, d z p	p	)	, the mean energy mean W	and the probability of forming inclusion complex
	inc P for the simulation of molecules with	ε =	0.006	eV at temperature	T =	293	K.
				σ (Å)	p d (Å)	p z (Å)	max P (%)	mean W	(eV)	inc P (%)
						2.4	1.80	-	0.5	2.7	-	2.28	4.1
						2.8	1.80	-	0.5	9.1	-	3.62	9.6
						3.0	1.00	-	0.5	25.1	-	4.23	12.1
						3.3	0.71	-	0.5	42.1	-	6.23	23.8
						3.7	0.50	-	0.5	68.7	-	9.89	32.6
						4.1	0.00	0.00	81.0	-	13.20	65.5

ACKNOWLEDGMENTS

We are grateful to the Ministerio de Educación y Ciencia and FEDER (FIS2005-02886), and the Ministerio de Ciencia e Innovación (FIS2009-0789) for their generous financial support.


Fabiana Zingone

Monica Siniscalchi

Pietro Capone

Raffaella Tortora

Paolo Andreozzi

Elisa Capone

Dr Carolina Ciacci
email: ciacci@unina.it

Quality of Sleep in Patients with Celiac Disease

Keywords: Coeliac disease < Disease-based, Small intestine < Organ-based, Psychiatric disorders sleep quality, quality of life, celiac disease 20 21

Methods: The participants were celiacs at diagnosis (C-N); celiacs on gluten free diet at 6 follow-up (C-T); and healthy volunteers (HV). Participants completed the Pittsburgh Sleep 7 Quality Index (PSQI), SF36, Zung and Fatigue scales, and State-Trait Anxiety Inventory 8 (STAI). 9

Results: The PSQI score was higher in C-N and C-T than in HV (p<0.001). A gluten-free 10 diet (GFD) did not improve the PSQI scores (p=0.245) in CD. The other test scores were 11 similar between celiacs at diagnosis and those on a GFD, while significant differences were 12 found between celiacs and volunteers. PSQI score was inversely associated with the quality 13 of the physical (r=-0.327 p=0.002) and mental (r=-0.455, p<0.001) components scores. [START_REF] Hadjivassiliou | Does cryptic gluten sensitivity play a part in neurological illness?[END_REF] The sleep quality scores were related to depression (r=0.633, p<0.001), fatigue (r=0.377, 15 p<0.001), state anxiety (r=0.484, p<0.001), and trait anxiety (r=0.467, p<0.001). 16 In the general population, increased stress, anxiety, depression and worry are associated 2 with poor subjective sleep quality [START_REF] Specchio | Insomnia, quality of life and psychopathological features[END_REF]. In addition, common medical problems are often 3 associated with abnormalities of sleep. In fact, patients with chronic medical disorders 4 often have fewer hours of sleep and less restorative sleep, as compared to healthy 5 individuals, and this poor sleep may worsen the subjective symptoms of the disorder [START_REF] James | Sleep related problems in common medical conditions[END_REF]. 6

Among gastrointestinal diseases, gastroesophageal reflux is a major cause of disrupted 7 sleep due to awakenings from heartburn, dyspepsia, coughing, or choking [START_REF] Kahrilas | Obstructive sleep apnea and reflux disease: bedfellows at best[END_REF]. Other 8 gastrointestinal diseases have not been studied. 9

Celiac disease is a chronic disease presenting with a broad spectrum of symptoms 10 and signs. Anxiety, depression, and other mood disorders are associated with celiac disease 11 [START_REF] Addolorato | State and trait anxiety and depression in patients affected by gastrointestinal diseases: psychometric evaluation of 1641 patients referred to an internal medicine outpatient setting[END_REF][START_REF] Siniscalchi | Fatigue in adult coeliac disease[END_REF][START_REF] Ludvigsson | Coeliac disease and risk of mood disorders-A general population-based cohort study[END_REF][START_REF] Addolorato | Anxiety but not depression decreases in coeliac patients after one-year gluten-free diet: a longitudinal study[END_REF]. More recently, restless legs syndrome has been shown to be frequent in celiac disease 12 (8). Most of the above-mentioned conditions may affect sleep. As the quality of sleep in 13 celiac disease has not been investigated systematically, this study investigated the quality of 14 sleep in a cohort of adult celiac patients. 15

16

MATERIALS AND METHODS 17

The study population consisted of adult celiacs consecutively recruited from September 18 The PSQI consists of 19 self-rated questions and five questions rated by a bed partner or 1 roommate (only the self-rated items are used in scoring the scale). The self-administered 2 portion consists of 15 multiple-choice items that inquire about the frequency of sleep 3 disturbances and subjective sleep quality, and four write-in items that inquire about typical 4 bedtime, wake-up time, sleep latency and sleep duration. The PSQI generates seven scores 5 that correspond to the domains listed previously. Each component score ranges from 0 (no 6 difficulty) to 3 (severe difficulty). The component scores are summed to produce a global 7 score (range 0-21). A PSQI global score >5 is suggestive of significant sleep disturbance 8 [START_REF] Buysse | The Pittsburgh Sleep Quality Index: A new instrument for psychiatric practice and research[END_REF][START_REF] Buysse | Quantification of subjective sleep quality in healthy elderly men and women using the Pittsburgh Sleep Quality Index[END_REF]. 9

To evaluate possible factors contributing to sleep quality, the SF-36 questionnaire, 10

Zung Self-rating Depression Scale, State-Trait Anxiety Inventory and a Fatigue visual 11 analogue scale (VAS), were administered. 12

SF-36 questionnaire 13

The SF-36 survey consists of a 36-item questionnaire that includes eight components: 14 physical functioning, role limitations due to physical health, bodily pain, general health, 15 vitality, social functioning, role limitations due to emotional health, and mental health. 16

These eight domains form two broader health dimension scales: the physical (PCS) and 17 mental (MCS) component scales. The SF-36 subscales and composite scores are presented 18 as means and standard deviations and range from 0 to 100, with higher scores indicating 19 better health and well-being. The PCS and MCS composite means and standard deviations 20 are 50±10 for the U.S. general population. PCS and MCS factors have been found to 21 account for 80~85% of the reliable variance in the eight SF-36 scales in patients, as well as 22 [START_REF] Ware | SF-36 physical and mental health summary scales: A user's manual[END_REF][START_REF] Ware | SF-36 health survey update[END_REF][START_REF] Ware | User's manual for the SF-36v2 health survey[END_REF]. Poor (low) scores on the PCS indicate limitations in physi-1 cal/role functions and general health, and bodily pain, while better (higher) scores suggest 2 no physical limitations, disabilities, or decrements in well-being. Similarly, low scores on 3 the MCS suggest frequent limitations in psychosocial health, emotional problems, and 4 reduced vitality (a fatigue construct), while high scores indicate frequent positive affect and 5 vitality, the absence of psychological distress, and reduced or no limitations in daily social 6 and role activities. 7

ZUNG Self-rating Depression Scale 8

The Zung Self-rating Depression Scale is a 20-item self-report questionnaire that is widely 9 used as a screening tool, covering affective, psychological, and somatic symptoms 10 associated with depression [START_REF] Hadjivassiliou | Does cryptic gluten sensitivity play a part in neurological illness?[END_REF][START_REF] Zung | From art to science[END_REF]. In this study, depressive symptoms were assessed by a 11 modified version of the Zung Self-rating Depression Scale (M-SDS), which contains 17/20 12 items belonging to the original version of the Zung scale [START_REF] Siniscalchi | Fatigue in adult coeliac disease[END_REF][START_REF] Addolorato | Anxiety but not depression decreases in coeliac patients after one-year gluten-free diet: a longitudinal study[END_REF]16). Three items for 13 gastroenterological symptoms of depression (decreased appetite, weight loss, and 14 constipation) were eliminated to avoid the possible bias due to CD. The cut-off score of the 15 M-SDS scale, for pathological depression is 44. 16

Fatigue visual analogue scale 17

This consisted of a single question: 'How did you feel during last week?' The patients were 18 asked to mark a visual analogue scale (VAS) consisting of a 10-cm line with 'I never feel 19 tired' at the left end and 'I always feel tired' at the opposite end. Therefore, the possible 20 score ranges from 0 to 10 (5). 21 Anxiety was assessed using the State-Trait Anxiety Inventory (STAI), which consists of 2 two axes (y1 for state anxiety and y2 for trait anxiety), both consisting of 20 multiple-3 choice items; the STAI can distinguish between existing anxiety and a predisposition to 4 anxious reactions as a personality characteristic, as previously described (17). This theory is 5 based on the conceptual distinction between anxiety as a transitory state and anxiety as a 6 relatively stable personality trait. State anxiety is conceptualised as an emotive state 7 characterised by subjective feelings perceived on a conscious level, such as apprehension 8 and tension, which varies with time; anxiety as a trait refers to individuals with an on-going 9 disposition towards anxiety (18). The subjects evaluated were grouped as high-and low-10 anxious and a value of 40 was used to distinguish between the two groups, according to 11 Spielberg et al. (19) and Weinstein (20). [START_REF] Ware | SF-36 health survey update[END_REF]

13

STATISTICAL ANALYSIS 14

Chi-square analysis and ANOVA were used for analyses on categorical and continuous 15 data, respectively. Continuous data were reported as means ± standard deviation (SD). 16 The study cohort consisted of three groups of 30 subjects each (10 males, 20 females). The 2 age at time of testing, education and marital status were similar among the three groups 3 (Table 1). The patients in C-T had been on a gluten-free diet for 6±5.9 years. 4

Nutritional indices were significantly different in C-N and C-T (blood hemoglobin: 5 11.67±0.87 g/dL and 12.3±0.52 g/dL respectively, p=0.002; plasma cholesterol: 6 165.6±13.47 mg/dL and 191.46±21.73 mg/dL respectively, p <0.001; serum albumin: 7 3.67±0.88 mg/dL and 4.09±0.37 mg/dL respectively, p <0.001; body mass index: 8 21.6±2.55 Kg/m 2 and 24.8±3.43 Kg/m 2 respectively, p= 0.007). 9

10

PSQI results 11

A progressive decrease of the score indicating pathological sleep, was found among 12 the three groups (p<0.001). The comparison between the two celiac groups indicated that 13 the PSQI score, did not differ between C-N and C-T (p>0.2) (Table 2). 14

The analysis of the single domains of the PSQI (subjective sleep quality, sleep 15 latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleep 16 medications, and daytime dysfunction) indicated a significant difference between celiac 17 patients (both C-N and C-T) and volunteers for each domain (p<0.05), except for sleep 18 duration, which was similar in the three groups (p=0.166). 19

SF36 results 20

The comparison of each single domain of the SF36 and the PCS and MCS values did not 21 show differences between celiacs at diagnosis and celiac on a gluten-free diet. The quality 22 VAS scores, and STAI y1 and y2 scale scores, as compared to volunteers. The results did 5 not significantly differ between untreated celiacs and celiacs on gluten free diet (Table 3). 6

Only two persons from C-N and two from C-T had a modified Zung score above the 7 threshold for major depression. Findings were similar after the exclusion of these patients 8 (C-N 5.92±3.52; C-T 4.82±2.14; HV 2.96±2.86; p= 0.001). 9

Nineteen persons from C-N, 16 from C-T, and 13 from HV scored above 40, for 10 STAI-y1 (p=0.3), scores more than 40 indicate an anxiety state; 17 persons from C-N, 15 11 from C-T, and 8 from HV scored above 40 for STAI-y2 (p=0.05), scores more than 40 12 indicate an anxiety trait. [START_REF] Ware | User's manual for the SF-36v2 health survey[END_REF] The PSQI score of all participants was inversely associated with the quality of the 14 physical (r=-0,327 p=0.002) and mental (r=-0.455, p<0.001) component scores, while 15 there were significant positive correlations between sleep quality and depression (Zung 16 r=0.633, p<0.001), fatigue (r=0.377, p<0.001), state anxiety (r=0.484, p<0.001), and trait 17 anxiety (r=0.467, p<0.001). 18

The age at testing was not related to sleep quality (r=0.105, p=0.323). In and should be considered a symptom of the disease. Sleep does not improve with a gluten-3 free diet, although the score tended to decrease, but did not reach those of the non-celiac 4 volunteers. In detail, in celiac patients, all items explored by the PSQI but one -sleep 5 duration-are impaired in comparison with volunteers. In celiac patients, sleep disorders 6 are directly related to depression, anxiety, and fatigue and inversely related to quality-of-7 life scale scores. Age, gender, education, marital status, and the presence of gastrointestinal 8 symptoms do not relate to the presence of sleep disorders. 9

Analysis of the SF36 questionnaire indicated that the scores on the PCS were low 10 before starting a gluten-free diet, indicating limitations in physical/role functions, bodily 11 pain, and general health, and these did not improve after treatment. The MCS score tended 12 to decrease on a gluten-free diet. As a low MCS score suggests frequent limitations in 13 psychosocial health, emotional problems, and reduced vitality (a fatigue construct), the data 14 suggest that living with the limitations imposed by a gluten-free diet induces a worsening 15 of psychosocial health and generates limitations in daily social and role activities. 16

This finding suggests that the sleep disorders are not related to the presence of 17 gastrointestinal symptoms at diagnosis, and is why they did not improve on the diet, in 18 contrast with the disappearance or improvement of most of the other symptoms. 19

The evaluation of the depression and anxiety scales scores in celiacs indicates that 20 celiacs are more anxious and depressed than volunteers. In celiacs, however, a gluten-free 21 diet did not induce any significant improvement in the score for any scale. This is in 22 anxiety, but not depression, after one year of a gluten-free diet. One possible explanation is 2 that celiacs from Addolorato study were evaluated after one year of gluten free diet. Our 3 celiacs have been evaluated after an average of six years from diagnosis. This different time 4 lapse might have played a role for the persistence (or renewal) of anxiety in our celiacs on 5 gluten free diet. 6

The present study has the limit of a comparison with general population controls. On the 7 other hand, a comparison with other chronic diseases is anyway unfeasible for the peculiar 8 characteristics of celiac disease, the limitations imposed by gluten free diet and the 9 disappearance of symptoms after some time from the beginning of the diet. However, the 10 study was designed to reduce the effect of this limitation. The PSQI was administered at 11 time of the first visit, hence when the person affering to the CD center had not yet received 12 any previous CD diagnosis. The selection of any other GI disease as control group would 13 have raised the problem of comparability because common GI diseases most often are with 14 a clinical presentation and a 'disease status' heavier that CD. [START_REF] Zung | From art to science[END_REF] Finally, the metabolic status of the treated group was much better that one of the untreated 16 group. Thus, the data indicate at least that sleep disorders in celiac disease are not fully 17 controlled after six-years of gluten-free diet despite of a substantial improvement of the 18 metabolic status. 19

In conclusion, our study confirms previous observations of a poor quality of life in 20 celiacs both before and after diet treatment. For the first time, our findings indicate that 21 sleep disorders are a frequent complaint in celiacs and should be added to the wide 22

  This study investigated the quality of sleep in CD. 5

Conclusions:

  Sleep disorders are common in CD also during treatment with GFD. Sleep 17 disorders are related to depression, anxiety and fatigue, and inversely related to quality of

  2009 to March 2010 from the Celiac Disease Centre of Federico II University (Naples, 19 Italy) and sex-and age-matched volunteers recruited from the hospital staff and friends of 20 CD patients. The study was approved by the Ethical Committee of the Federico II 21 University of Naples (Diagnosis and Follow-up of Celiac Disease n10/2003-2013). 22 divided into two groups: celiac patients at diagnosis on gluten-containing 1 diet (celiac-new, C-N) and celiac patients at follow-up on gluten free diet, since at least one 2 year and with negative anti-transglutaminase IgA antibody (celiac-treated, C-T). 3 Questionnaires were administered in the morning of the annual visit which is required after 4 CD diagnosis for coverage the costs of gluten free food by the Italian National Health 5 System. None of patients refused to answer to this questionnaire. For all celiac patients, the 6 diagnosis was based on the presence of anti-transglutaminase IgA and anti-endomysium 7 antibodies and an intestinal biopsy compatible with gluten-related damage. The control 8 group (healthy volunteer, HV) consisted of individuals who were tested and found negative 9 for serum markers of celiac disease. . 10 Data were collected on age, gender, marital status, education and laboratory nutritional 11 indices. The inclusion criteria of the study cohort were written informed consent, age 19 to 12 60 years, absence of major psychiatric disease, absence of cancer, and no pregnancy or 13 children below 3 years old that may affect the quality and modality of sleep. 14 15 Pittsburgh Sleep Quality Index 16 All participants completed the Pittsburgh Sleep Quality Index (PSQI) to evaluate 17 sleep quality. The PSQI was developed to measure sleep quality during the previous month 18 and to discriminate between good and poor sleepers. Sleep quality is a complex 19 phenomenon that involves several dimensions, each of which is covered by the PSQI. The 20 domains covered include subjective sleep quality, sleep latency, sleep duration, habitual 21 sleep efficiency, sleep disturbances, the use of sleep medications, and daytime dysfunction. 22

  Categorical data were given as counts and percentages. The Spearman correlation 17 coefficient was used to test for correlations between sleep quality and quality of life scores, 18 depression scale, fatigue severity, STAI scores, and age. The Statistical Package for Social 19 Sciences (SPSS) 13.0 was used to analyze the data. A p value ≤ 0.

  CD patients, (C-N and C-T) was significantly lower than that of the volunteers for 1 each domain (Figure1). 2Zung, fatigue, and anxiety scale results 3Celiac patients showed a significant difference in the modified Zung scale scores, fatigue 4

1

 1 addition, 19 gender (p=0.295), education (p=0.307), marital status (p=0.223), and gastrointestinal 20 symptoms (p=0.403) were not related to sleep quality. Data from this study indicated that sleep disorders are common in untreated celiac disease 2

  study of Addolorato, which found a significant improvement in state 1

3 gluten

 3 and signs of CD. Studies should determine if medical therapy that 1 improves sleep also improves the quality of life of celiac patients on a gluten-free diet. 2 Longitudinal studies are needed to confirm the present findings and to assess the effect of C, Iavarone A, Mazzacca G, De Rosa A. Depressive symptoms in adult celiac disease. Scand J Gastroenterol. 1998 Mar;33(3):247-50. 17. Peterson R.A. and Reiss R.L. (1987). The anxiety sensitivity index: Construct validity and factor anal analytic structure. Journal of Anxiety Disorders. (1), 265-277. 18. Spielberger C. D., Gorsuch R.L., and Lushene. R.E. (1970

Figure 1 . 5 (

 15 Figure 1. The mean±SD of the eight component scores of the SF36 questionnaire (physical 1 functioning, role limitations due to physical health, bodily pain, general health, vitality, 2 social functioning, role limitations due to emotional health, and mental health) and the 3 mean±SD of the physical (PCS) and mental (MCS) component scales in celiac patients at 4 diagnosis (celiac-new, C-N), on a gluten-free diet (celiac-treated, C-T), and volunteers 5 (healthy volunteer, HV). 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Table 1 .

 1 ). Manual for the State-Trait Anxiety Inventory. Palo Alto, CA: Consulting Psychologists Press 19. Spielberger C. D., Reheiser E.C., Ritterband L.M., Sydeman, S.J., and Unger, K.K. Assessment of Emotional States and Personality Traits: Measuring Psychological Vital Signs. In Butcher, J.N. (Ed.) Clinical Personality Assessment: Practical Approaches. New York: Oxford University Press, 1995. Demographics of the study population (celiac-new C-N, celiac-treated C-T, 1

	20. Weinstein AM. Visual ERPs evidence for enhanced processing of threatening
	information in anxious university students. Biol Psychiatry. 1995 Jun
	15;37(12):847-58.
	1
	2
	3
	4
	5
	6
	7
	8

Table 2 .

 2 The percentage of scores indicating pathological sleep and the mean±SD of the 1 PSQI results for celiac patients at diagnosis (celiac-new, C-N), on a gluten-free diet (celiac-2

	Page 17 of 21		Alimentary Pharmacology & Therapeutic
	3	treated, C-T), and volunteer (healthy volunteer, HV).
			C-N	C-T	HV	p
		%PSQI pathological (>5)	50%	33.3%	23.33%	0.093
		PSQI in the three groups	6.20±3.64	5.23±2.64	2.96±2.86	<0.001
	4

Table 3 .

 3 The mean±SD of the scores of the modified Zung scale for depression, the VAS (1 1 to 10) for fatigue, and STAI-y1 and -y2 for state and trait anxiety in the study population 2

	3	(celiac-new C-N, celiac-treated C-T, healthy volunteer HV)
	4
			C-N	C-T	HV	p
		Modified Zung	34.23±6.73	31.40±7.19	25.40±5.85	<0.001
		Fatigue VAS	6.13±2.54	5.5±2.17	3.43±2.36	<0.001
		STAI-y1	46.23±10.70	43.90±10.95	37.70±8.67	0.005
		STAI-y2	42.90±8.10	42.73±9.55	36.23±8.02	0.004


Marina Aloi

Giovanni Di Nardo

Francesca Conte

Luigi Mazzeo

Nelson Cavallari

Federica Nuti

MD Salvatore Cucchiara
email: salvatore.cucchiara@uniroma1.it

Laura Stronati

METHOTREXATE IN PEDIATRIC ULCERATIVE COLITIS: A RETROSPECTIVE SURVEY AT A SINGLE TERTIARY REFERRAL CENTER

Keywords: Inflammatory bowel disease < Disease-based, Ulcerative colitis < Disease-based, Paediatric gastroenterology < Topics, Immunosuppression

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Inflammatory bowel diseases (IBD), i.e. ulcerative colitis (UC) and Crohn's disease (CD), are chronic inflammatory disorders of the gut characterized by recurrent exacerbations intermingled with phases of quiescence [START_REF] Grossman | Specific considerations in the treatment of pediatric inflammatory bowel disease[END_REF][START_REF] Sauer | Pediatric inflammatory bowel disease: highlighting pediatric differences in IBD[END_REF]. Most patients with active UC respond to aminosalicylates (5-ASA) or CS. The latter are highly effective in inducing remission, however, a considerable group of patients relapse when steroids are withdrawn [START_REF] Hyams | The natural history of corticosteroid therapy for ulcerative colitis in children[END_REF].

Moreover, children on long term CS exhibit serious adverse effects, among which linear growth delay, osteopenia, and infections are the most cumbersome [START_REF] Hyams | The natural history of corticosteroid therapy for ulcerative colitis in children[END_REF][START_REF] Leonard | Glucocorticoid-induced osteoporosis in children: impact of the underlying disease[END_REF]. CS sparing while maintaining remission is a tricky therapeutic strategy in the management of IBD: for this purpose IMs have successfully been introduced into the therapeutic armamentarium of this disorder [START_REF] Rufo | Current therapy of inflammatory bowel disease in children[END_REF][START_REF] Punati | Effect of early immunomodulator use in moderate to severe pediatric Crohn's disease[END_REF]. The most commonly used IMs for IBD are thiopurine analogs such as azathioprine (AZA) and 6-mercaptopurine (6-MP) [START_REF] Braus | Advances in the pathogenesis and treatment of IBD[END_REF]: however, serious adverse events may lead to cessation of therapy in up to 25% of patients, while unresponsiveness occurs in 15-20% of them [START_REF] Homan | Managing complicated Crohn's disease in children and adolescents[END_REF][START_REF] Akoberg | Crohn's disease: current treatment options[END_REF]. Thiopurine intolerant or unresponsive patients are usually treated with alternative IMs or biological therapy. Recently, there has been a great concern due to a reported increase in the risk of lymphoma in IBD patients chronically treated with AZA [START_REF] Kandiel | Increased risk of lymphoma among inflammatory bowel disease patients treated with azathioprine and 6-mercaptopurine[END_REF]. The chronic use of thiopurine analogs has been also challenged following publication of cases of hepato-splenic T cell lymphoma in young adults receiving them in association with biologics [START_REF] Cucchiara | Pediatric inflammatory bowel disease and risk of lymphoma: should we revise our treatment strategies?[END_REF]. Thus, pediatric gastroenterologists not uncommonly consider alternative IMs. Methotrexate (MTX), a potent folic acid antagonist, causes a decrease in purine production at the cellular level [START_REF] Jolivet | The pharmacology and clinical use of methotrexate[END_REF][START_REF] Cronstein | The mechanism of action of methotrexate[END_REF]. A direct antiinflammatory effect of MTX (i.e. inhibition of cellular proliferation, decrease in the formation of antibodies and in the production of inflammatory mediators, induction of apoptosis) has also been demonstrated [START_REF] Cronstein | The mechanism of action of methotrexate[END_REF] leading to its use in several inflammatory disorders, such as psoriasis and rheumatoid arthritis [START_REF] Feely | Therapeutic options for rheumatoid arthritis[END_REF][START_REF] Kalb | Methotrexate and psoriasis: 2009 National Psoriasis Foundation Consensus Conference[END_REF]. However, the role of MTX in IBD remains somewhat unclear, although it has been shown to be effective both for induction and maintenance of remission in adults with CD [START_REF] Kozarek | Methotrexate induces clinical and hystologic remission in patients with refractory inflammatory bowel disease[END_REF][START_REF] Feagan | Methotrexate in the treatment of Crohn's disease[END_REF][START_REF] El-Matary | Methotrexate for maintenance of remission in ulcerative colitis[END_REF]. While very few reports have shown the effectiveness and safety of MTX in children with CD [START_REF] Uhlen | Efficacy of Methotrexate in pediatric Crohn's disease: a french multicenter study[END_REF][START_REF] Weiss | Methotrexate treatment in pediatric Crohn's disease patients intolerant or resistant to purine analogues[END_REF], reports in UC are lacking and confined to adult patients [START_REF] Oren | Methotrexate in chronic ulcerative colitis: a double blind, randomised, Israeli multicentre trial[END_REF][START_REF] Patel | Methotrexate for maintenance of remission in Crohn's disease[END_REF][START_REF] Paoluzi | Azathioprine or methotrexate in the treatment of patients with steroid dependent or steroid resistant ulcerative colitis: results o fan open-label study on efficacy and tolerability in inducing and maintaining remission[END_REF]. Thus, the aim of this study was to report the experience of a tertiary referral Center for Pediatric Gastroenterology in the use of MTX in children with UC.

PATIENTS AND METHODS

This is a retrospective longitudinal study conducted at a single tertiary referral Center for Pediatric Gastroenterology. The Department database was screened for all children diagnosed with UC by 2004, and those treated with MTX were identified. Diagnosis of UC was based on widely agreed endoscopic and histological criteria [START_REF] Justinich | Inflammmatory bowel disease in children and adolescence[END_REF], after excluding infectious and systemic disease, food allergies and malabsorption syndromes. All patients underwent ileocolonoscopy with a pediatric videocolonoscope (Olympus, PCF Q 180 I) following conscious sedation with i.v. pethidine (1-2 mg/Kg) and midazolam (0.1 mg/Kg) or under general anesthesia. Endoscopic criteria for UC were diffuse continuous inflammation extending from the rectum proximally and characterized by granularity, friability, small diffuse superficial ulcers superimposed on a background of loss of vascular mucosal pattern and normal appearing distal ileal mucosa. Histological features for UC were a continuous intense mixed acute and chronic inflammatory infiltrate of the lamina propria, distortion of crypt architecture and/or crypt atrophy, basal plasmacytosis, cryptitis and crypt abscesses, Paneth cell metaplasia distal to the ascending colon. The behavior of disease activity, measured through PUCAI [START_REF] Turner | Development, validation and evaluation of a Pediatric Ulcerative Colitis Activity Index: a Prospective Multicenter Study[END_REF] and the use of corticosteroids (evaluated baseline and at 3, 6,12 months post-MTX) were the main outcomes. PUCAI is a validated, noninvasive, multi-item measure of disease activity with established cut-off values for remission (<10 points), mild (10-34 points), moderate (35-64 points), and severe disease (65-85 points), developed in 2007 [START_REF] Turner | Development, validation and evaluation of a Pediatric Ulcerative Colitis Activity Index: a Prospective Multicenter Study[END_REF][START_REF] Turner | A systematic prospective comparison of noninvasive disease activity indices in ulcerative colitis[END_REF][START_REF] Turner | Appraisal of the pediatric ulcerative colitis activity index (PUCAI)[END_REF]. Clinical remission was defined as a PUCAI [START_REF] Turner | Development, validation and evaluation of a Pediatric Ulcerative Colitis Activity Index: a Prospective Multicenter Study[END_REF]. Medical records of patients evaluated before 2007 were retrospectively reviewed by an experienced physician unaware of medical therapy and PUCAI sub-domain scores were attributed. The use of CS, adverse effects and surgical procedures during the study period were the secondary outcomes recorded. Thiopurine intolerance was defined as a requirement to discontinue thiopurine because of side effects. Thiopurine refractoriness was defined as no clinical improvement to a trial of at least 12 weeks therapy. MTX was given weekly by intramuscular injection at the dose of 15 mg/m² body surface area. All children received 5 mg of intramuscular folic acid weekly, 6 hours after MTX. To detect potential hepatic, renal or haematological side effects of MTX therapy, patients were monitored every 2 weeks during the first 2 months and every 8 to 12 weeks thereafter [START_REF] Kremer | Methotrexate for rheumatoid arthritis: suggested guidelines for monitoring liver toxicity. American College of Rheumatology[END_REF]. Laboratory tests included full blood count, inflammatory, renal and liver function parameters, serum albumin, electrolytes, lipase and amylase. Liver ultrasound was performed if liver function tests were abnormal, while chest x-ray was performed in all patients before starting MTX to rule out pulmonary fibrosis.

Statistical analysis for the comparison of the qualitative variables was carried out by χ² test or Fisher's exact test (where appropriate). Continuous variables were compared using the Mann-Whitney nonparametric U test. A p value less than or equal to 0.05 was considered as significant. Data were given as median (and ranges) and as mean ±SD.

RESULTS

Of the 38 children with active UC treated by MTX with a follow-up of at least 12 months, 32 ( MTX, an extensive colitis (pancolitis) was present in 24 (75%), a left-sided colitis in 5 (16%), and a proctitis in 3 (8%). AZA had been introduced in all but 4 patients as first-line IM agent. Thiopurine therapy was discontinued because of primary nonresponse or relapse in 18 patients (56%), and intolerance/toxicity in 10 (31%). In 4 patients MTX had been introduced as the first-line IM because of coexisting joint disease (spondyloarthropathy). MTX was administered parenterally in all patients with a mean dose of MTX for patient of 13.7±3.6 mg/m²/week. All patients were on chronic treatment with mesalazine (75-100 mg/Kg/day) that was maintained in addition to MTX during all the observation period.

Of the 32 children on MTX a clinical improvement (response or remission) was seen in 72%, 63%, and 50% at 3, 6, and 12 months respectively (Figure ): this included 6 of 10 with AZA intolerance or toxicity, and 14 of 18 with AZA refractoriness. Remission was observed in 10 patients at 3 months, and in 9 at both 6 and 12 months; response was observed in 13, 11 and 7 patients at 3, 6, and 12 months, respectively. Mean PUCAI 6 months before the introduction of MTX was 40.5±23.6 (moderate disease), 49.5±23.3 at the introduction of MTX, and 32.9±21.9, 29.5±22.8, 29.4±25.9 (mild disease) at 3, 6 and 12 months post-MTX, respectively (p: 0.03). At the beginning of MTX 16 patients were receiving oral prednisolone with a mean dose of 1.24 mg/Kg/day (range 0.2-3.2 mg): tapering of CS was possible in 14 (87.5%), with complete discontinuation in 11 (68.7%) within the first 3 months, and in other 2 patients after 6 months of MTX. At 12 months, 8 children were receiving short courses of CS because of disease relapse. At the end of the study period 8 patients had a sustained steroid-free clinical remission.

No surgery was performed during the observation period. There were adverse reactions in 8 patients within the first 3 months of therapy: asymptomatic elevation of alanine aminotransferase (ALT 3 to 4 times the upper limit of normal) in 4, with normalization in all after dose reduction of MTX, whereas 4 needed to discontinue the drug because of

DISCUSSION

The natural history of UC is characterized by phases of remission intermingled with recurrent flare-ups, that occur in an unpredictable fashion and impair the quality of life of the patients. The main goals in the management of pediatric UC are to control disease exacerbations and avoid prolonged use of CS [START_REF] Grossman | Specific considerations in the treatment of pediatric inflammatory bowel disease[END_REF]. The classical medical armamentarium for UC includes anti-inflammatory drugs, CS and IMs. While CS have a short-term clinical efficacy, they do not change the disease natural history and cause a number of troublesome side effects. The IMs are considered as steroid sparing agents, when a longterm remission is attainable [START_REF] Markowitz | Immunomodulatory therapy for pediatric inflammatory bowel disease: changing patterns of use, 1990-2000[END_REF]. Although thiopurines are the most commonly used IMs, some patients exhibit unresponsiveness or intolerance to them and MTX has been proposed as an alternative immunomodulatory therapy [START_REF] Grossman | Specific considerations in the treatment of pediatric inflammatory bowel disease[END_REF][START_REF] Sauer | Pediatric inflammatory bowel disease: highlighting pediatric differences in IBD[END_REF]. Whereas there are few retrospective reports on MTX in patients with CD, there are no data on the effectiveness and safety of MTX in pediatric UC. Our study shows that children with UC may benefit from MTX, both to induce and to mantain remission, as well as in reducing CS exposure.

Of the 32 treated patients, we observed an overall clinical improvement in 23, 20 and 16 at 3, 6 and 12 months, respectively. Comparable high response rates are reported in adult studies. A recent retrospective study in 32 adults nonresponsive or intolerant to thiopurines reported a clinical response of 68% using either oral or parenteral MTX at an induction dose of 25 mg/week [START_REF] Wahed | Efficacy of methotrexate in Crohn's disease and ulcerative colitis patients unresponsive or intolerant to azathioprine/mercaptopurine[END_REF]. Previously, a response rate of 72% and a remission rate of 42% had been reported in adults with active UC formerly treated with thiopurines [START_REF] Paoluzi | Azathioprine or methotrexate in the treatment of patients with steroid dependent or steroid resistant ulcerative colitis: results o fan open-label study on efficacy and tolerability in inducing and maintaining remission[END_REF]. The evidence for using MTX in UC is restricted to observational studies, while the only randomized, placebo-controlled trial in adults found no efficacy, however it was underpowered and a low oral dose of MTX (12.5 mg weekly) was used [START_REF] Oren | Methotrexate in chronic ulcerative colitis: a double blind, randomised, Israeli multicentre trial[END_REF]. In our study the majority of patients started MTX during a flare-up of the disease, thus it is conceivable that a "regression to the mean phenomenon" might contributed to the clinical response to the therapy, leading to a lower disease activity just by chance. However, as shown by longitudinal assessment of disease activity, our patients had highly active disease already six months before starting MTX (i.e. medium PUCAI 40.5±23.6), thus reducing the likely impact of this phenomenon.

We gave MTX intramuscularly since it has previous shown that this way of administration may be more useful due to the variable oral bioavailability of the drug [START_REF] Cummings | Oral methotrexate in ulcerative colitis[END_REF]. Subcutaneous injection is as an alternative method of parenteral administration: as compared to intramuscular injection it produces similar pharmacokinetics, and leads to greater patient comfort by reducing local complication rate at the injection site [START_REF] Patel | Methotrexate for maintenance of remission in Crohn's disease[END_REF]. The majority of our patients improving on MTX achieved clinical remission within 10-12 weeks (range, [START_REF] Punati | Effect of early immunomodulator use in moderate to severe pediatric Crohn's disease[END_REF][START_REF] Braus | Advances in the pathogenesis and treatment of IBD[END_REF][START_REF] Homan | Managing complicated Crohn's disease in children and adolescents[END_REF][START_REF] Akoberg | Crohn's disease: current treatment options[END_REF][START_REF] Kandiel | Increased risk of lymphoma among inflammatory bowel disease patients treated with azathioprine and 6-mercaptopurine[END_REF][START_REF] Cucchiara | Pediatric inflammatory bowel disease and risk of lymphoma: should we revise our treatment strategies?[END_REF][START_REF] Jolivet | The pharmacology and clinical use of methotrexate[END_REF], this indicating that MTX is clinically efficacious earlier than thiopurines, which have a delayed onset of action, usually 2 or 3 months after commencement of administration [START_REF] Markowitz | Immunomodulatory therapy for pediatric inflammatory bowel disease: changing patterns of use, 1990-2000[END_REF].

The anti-inflammatory effect of MTX at low-doses has not clearly been defined. Whereas MTX at high doses has an antiproliferative and cytotoxic effect, by inhibiting the enzyme dihydrofolate reductase and leading to defective DNA synthesis and cell death [START_REF] Jolivet | The pharmacology and clinical use of methotrexate[END_REF], at lowdoses it acts mainly as an IM [START_REF] Ravikumara | Role of methotrexate in the management of Crohn's disease[END_REF]. The immunomodulatory effect of MTX is poorly understood but it involves increased concentrations of adenosine [START_REF] Morabito | Methotrexate and sulfasalazine promote adenosine release by a mechanism that requires ecto-5'-nucleotidasemediated conversion of adenine nucleotides[END_REF], inhibition of cellular proliferation and induction of apoptosis [START_REF] Genestier | Immunosuppressive properties of methotrexate: apoptosis and clonal deletion of activated peripheral T cells[END_REF], and decreased production of inflammatory mediators, such as interleukins and eicosanoids [START_REF] Cronstein | The mechanism of action of methotrexate[END_REF].

The safety of MTX has been extensively studied in patients with chronic inflammatory diseases such as rheumatoid arthritis and psoriasis. In the short term, the most common side effects are of gastrointestinal type, in particular nausea, vomiting and diarrhea, and asymptomatic elevation of transaminases. Longer-term safety concerns include bone marrow suppression, opportunistic infection due to immune suppression, pneumonitis, [START_REF] Markowitz | Immunomodulatory therapy for pediatric inflammatory bowel disease: changing patterns of use, 1990-2000[END_REF]. The supplementation of folates during MTX therapy seems to reduce the risk of cytopenia and the severity of gastrointestinal symptoms without reduction in efficacy and bioavailability of the drug [START_REF] Nathan | A signle center experience of methotrexate in the treatment of Crohn's disease and ulcerative colitis: a case for subcutaneous administration[END_REF]. The majority of our patients preferred folic acid intramuscularly despite the latter is widely know to be very effective by oral route [START_REF] Pavy | Methotrexate therapy for rheumatoid arthritis: clinical practice guidelines based on published evidence and expert opinion[END_REF].

In our study MTX was well tolerated and safe. Four children needed to discontinue the drug due to intractable nausea unresponsive to folic acid and antiemetics; in 4 patients there was a transient abnormality in liver function tests, that resolved with MTX dose reduction. Due to the rarity of severe chronic liver disease related to MTX therapy and according to recent recommendations [START_REF] Aberra | Monitoring of inflammatory bowel disease[END_REF][START_REF] Siegel | Review article: practical management of inflammatory bowel disease patients taking immunomodulators[END_REF] a conservative approach was done and no patient underwent liver biopsy.

It is still unsettled whether MTX may be considered as a first-line IM agent in the treatment of UC. Indeed, there are no head-to-head studies comparing MTX and AZA in IBD and no data are available on MTX as first-line therapy while its is commonly reported in patients unresponsive or intolerant to thiopurines. In our study only 4 patients with UC and spondyloarthopathy received MTX as first-line IM agent: they improved both in intestinal and articular symptoms without adverse reactions.

Although this study has some limitations such as the small sample size, the lack of controls and the retrospective nature, however it suggests that MTX can be a useful agent for the management of pediatric UC patients, unresponsive or intolerant to thiopurines. Large, controlled prospective trials are warranted to better define the role and the place of MTX in the treatment of pediatric UC.

  and clinical response as a decrease in the PUCAI of at least 20 points as compared to baseline

  No haematological or infectious complications were observed in this case series.

Figure .

 . Figure. Rate of clinical remission/response and relapse/intolerance to MTX in pts with UC over the 1year study period 254x190mm (72 x 72 DPI)

  16 males) provided complete data on all items of the PUCAI with a valid total score (median age: 14.0 years; range: 8.3-19.8; IQR 12.5). The mean age at diagnosis of UC was 10±4.3 years, and MTX was commenced 23.4±12.3 months after diagnosis. The Table summarizes baseline clinical characteristics of the patients. At the introduction of

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