MD¹ Birgit Abler
email: birgit.abler@uni-ulm.de

MD² Angela Seeringer

Antonie Hartmann

PhD¹ Georg Grön

MD³ Coraline Metzger

MD³ Martin Walter

MD² Julia Carolin Stingl

Antonie Hartmann¹

Dr Birgit Pd

MD Abler

Al

Neural correlates of antidepressant-related sexual dysfunction -a placebo-controlled fMRI study on healthy males under subchronic paroxetine and bupropion

Keywords: Bupropion, Paroxetine, erotic stimulation, sexual dysfunction, fMRI

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INTRODUCTION

Selective serotonin reuptake inhibitors (SSRIs) are the most widely prescribed antidepressant drugs [START_REF] Bauer | Prescribing patterns of antidepressants in Europe: Results from the factors influencing depression endpoints research (FINDER) study[END_REF]. For the majority of relevant diagnoses, regular intake is recommended for months or even longer [START_REF] Gaebel | Praxisleitlinien in Psychiatrie und Psychotherapie Bd. 5: Behandlungsleitlinie Affektive Erkrankungen[END_REF].

However, despite the positive profile of SSRIs, premature discontinuation is common and frequently discussed to account for non-response and relapse [START_REF] Bull | Discontinuing or switching selective serotonin-reuptake inhibitors[END_REF].

Sexual dysfunction is a typical side effect of SSRI treatment [START_REF] Serretti | Treatment-emergent sexual dysfunction related to antidepressants: a meta-analysis[END_REF]) that conditions discontinuation and may represent the most clinically relevant side effect among patients in their twenties or thirties [START_REF] Bishop | Serotonin 2A -depression and SSRI-associated sexual side-effects[END_REF]. Decreased sexual desire and interest in sexual activity along with erectile dysfunction is reported in up to 60% of treatments with SSRIs [START_REF] Gregorian | Antidepressant-induced sexual dysfunction[END_REF] with the highest rates of up to 64% observed under paroxetine [START_REF] Clayton | Prevalence of sexual dysfunction among newer antidepressants[END_REF]. Since sexual dysfunction is similarly reported under treatment with dual antidepressants inhibiting serotonin and norepinephrine reuptake [START_REF] Montejo | Incidence of sexual dysfunction associated with antidepressant agents: a prospective multicenter study of 1022 outpatients. Spanish Working Group for the Study of Psychotropic-Related Sexual Dysfunction[END_REF], but not under selective norepinephrine reuptake inhibitors [START_REF] Clayton | Lack of sexual dysfunction with the selective noradrenaline reuptake inhibitor reboxetine during treatment for major depressive disorder[END_REF], the serotonergic component seems to represent the crucial part in mediating sexual dysfunction.

Bupropion is a selective dopamine and norepinephrine reuptake inhibitor and is named as an alternative which is reportedly not associated with side effects on sexual functioning [START_REF] Coleman | A placebo-controlled comparison of the effects on sexual functioning of bupropion sustained release and fluoxetine[END_REF]. Bupropion has even been suggested as a potential treatment for SSRI-induced sexual dysfunction or hypoactive sexual desire disorder in women [START_REF] Modell | Effect of bupropion-SR on orgasmic dysfunction in nondepressed subjects: a pilot study[END_REF]. This is in line with clinical observations relating dopamine agonists to hypersexuality [START_REF] Voon | Chronic dopaminergic stimulation in Parkinson's disease: from dyskinesias to impulse control disorders[END_REF] and dopamine antagonists to sexual dysfunction (Baldwin and Mayer 2003). Animal experiments clearly support the notion that modulation of central, but not peripheral serotonergic pathways is involved in SSRI-related alteration of sexual functioning [START_REF] Waldinger | Premature ejaculation and serotonergic antidepressants-induced delayed ejaculation: the involvement of the serotonergic system[END_REF]. Enhancement of central serotonergic neurotransmission with stronger activation of inhibitory postsynaptic autoreceptors [START_REF] Blier | Long-term 5-HT reuptake blockade, but not monoamine oxidase inhibition, decreases the function of terminal 5-HT autoreceptors: an electrophysiological study in the rat brain[END_REF] has been postulated as a likely SSRI effect modulating the inhibitory control of ejaculation [START_REF] Giuliano | Serotonin and premature ejaculation: from physiology to patient management[END_REF]. However, the actual pathophysiology of SSRI related sexual dysfunction is poorly understood. Using functional magnetic resonance imaging (fMRI), we investigated the neural correlates of erotic stimulation under paroxetine. Because sexual dysfunction including decreased libido and anorgasmia is a common symptom of depression irrespective of antidepressant treatment, we investigated healthy subjects under subchronic treatment. To control for the effects of serotonergic versus dopaminergic/noradrenergic antidepressant treatment, we set up a randomized cross-over design with each subject receiving either paroxetine, or bupropion or placebo over the course of one week separated by sufficient washout phases. Stimulation with erotic video clips has already been demonstrated as a reliable tool to investigate brain regions related to sexual functioning [START_REF] Redoute | Brain processing of visual sexual stimuli in human males[END_REF] and to contrast medication effects on these brain regions in patients suffering from depression [START_REF] Kim | Treatment with Selective Serotonin Reuptake Inhibitors and Mirtazapine Results in Diefferential Brain Activation by Visual Erotic Stimuli in Patients with Major Depressive Disorder[END_REF]. Under placebo, we expected to find activation in those brain regions reported to be active during erotic stimulation according to previous fMRI studies, i.e. the anterior cingulate cortex, lateral occipital cortex, insula, amygdala, hypothalamus, thalamus and the ventral striatum [START_REF] Arnow | Brain activation and sexual arousal in healthy, heterosexual males[END_REF][START_REF] Ferretti | Dynamics of male sexual arousal: distinct components of brain activation revealed by fMRI[END_REF][START_REF] Moulier | Neuroanatomical correlates of penile erection evoked by photographic stimuli in human males[END_REF][START_REF] Mouras | Identifying distinct components in the cerebral treatment of visual sexual information through functional neuroimaging[END_REF][START_REF] Redoute | Brain processing of visual sexual stimuli in human males[END_REF]Walter, et al 2008a;[START_REF] Walter | High resolution fMRI of subcortical regions during visual erotic stimulation at 7 T[END_REF]. In line with previous studies of blunted ventral striatal activation under SSRIs [START_REF] Mccabe | Diminished neural processing of aversive and rewarding stimuli during selective serotonin reuptake inhibitor treatment[END_REF], paroxetine was expected to exert inhibitory effects on reward system activation, the latter being identified as specifically elicited by erotic stimulation as a primary reward (Walter, et al 2008a). By contrast, bupropion, as a dopamine reuptake inhibitor, was expected to enhance activation of the dopaminergic reward system as reported for the administration of Ldopa or dopamine agonists in healthy subjects [START_REF] Pessiglione | Dopaminedependent prediction errors underpin reward-seeking behaviour in humans[END_REF]. SSRI-related attenuation of ventral anterior cingulate cortex (ACC) and insula was expected from recent findings of similar effects of subchronic administration of citalopram on emotional anticipation in healthy subjects [START_REF] Simmons | Subchronic SSRI administration reduces insula response during affective anticipation in healthy volunteers[END_REF].

MATERIALS AND METHODS

Subjects: 23 healthy male, heterosexual subjects (3 left-handed) were randomized to take placebo, bupropion and paroxetine at routine clinical doses for seven days each in a counterbalanced order. After each medication period these subjects were scheduled to take part in the fMRI experiment. From this sample, 18 subjects aged 23-34 years (mean 25.4, SD 2.9, 3 left-handed) completed all three fMRI sessions (see figure 1) and were therefore included in the final analyses. Two of the five subjects who did not enter the final analyses were excluded because data from one of the three fMRI session could not be considered due to technical problems (spiking, coil malfunction). Two further subjects were excluded from the analyses because blood levels of paroxetine were too low to be quantified rendering intake of the drug doubtful. One subject had to be excluded because the blood sample for the bupropion levels was lost. The mean plasma bupropion-level of included subjects was 59.9 ng/ml (SD 27.7), the mean plasma paroxetine-level was 28.1 ng/ml (SD 17.9). Blood samples were taken after each fMRI session, that is about three hours after drug intake. Plasma levels were assessed as an estimate of drug exposure and drug adherence. More precise pharmacological profiles would have meant taking more blood samples which, however, was not compatible with the present study design. Blood levels were not analyzed before completion of the whole study.

The study was approved by the local ethical review board of the University of Ulm. All volunteers gave written informed consent prior to the study in accordance with the Declaration of Helsinki. Each participant received a full medical evaluation including a medical history, a physical examination, and a Structured Clinical Interview for DSM-IV Axis I Psychiatric Disorders (SCID) as well as laboratory blood tests and an electrocardiogram to exclude cardiac, renal or hepatic preconditions. Exclusion criteria were any current or past psychiatric or neurological illnesses, any serious general medical condition, use of illegal drugs and excessive consumption of caffeine or alcohol (>14 units/week). Subjects were also excluded for any significant baseline sexual dysfunction or sexual disorders (see supplementary materials for details). One subject took a single dose of acetaminophen, one a single dose of metamizole and one used a steroid inhaler for mild allergic asthma parallel to the study medication.

Other than that, none of the subjects took any medication and none had taken any other medication within the week before the study. Subjects were asked to refrain from alcohol parallel to the study medication and from coffee on the day of the scans.

Upon recruitment, subjects completed two self-report scales, the Center for Epidemiologic Studies Depression Scale, CES-D [START_REF] Radloff | The CES-D scale: A self-report depression scale for research in the general population[END_REF], German version: Allgemeine Depressions Skala/ADS, [START_REF] Hautzinger | Allgemeine Depressionsskala[END_REF] and a German version [START_REF] Reinecke | Sexuelle Dysfunktionen bei Patienten einer verhaltenstherapeutischen Hochschulambulanz: Häufigkeit, Erkennen, Behandlung[END_REF] of the Massachusetts General Hospital Sexual Function Questionnaire, MGH-SFQ [START_REF] Labbate | Sexual Dysfunction in Male Psychiatric Outpatients: Validity of the Massachusetts General Hospital Sexual Functioning Questionnaire[END_REF]. The MGH-SFQ is based on self-report and assesses sexual interest, sexual arousal, ability to achieve orgasm, ability to achieve and maintain an erection, and overall sexual satisfaction over the past month. The questionnaire was modified to reflect subjective sexual dysfunction only over the past week of medication.

Study design and procedures:

The entire study was performed as a randomized double-blind placebo-controlled within-subject cross-over design (figure 1). Each subject was investigated on three different occasions, once under placebo (PLAC: gelatine capsule filled with mannitol powder), once under paroxetine (PAR: 20 mg, identical capsule with pulverized paroxetine) and once under bupropion (BUP: 150 mg, identical capsule with bupropion) at intervals of at least fourteen days washouttime. For each treatment subjects were instructed to take one capsule each morning for seven days to reach steady state blood levels. SSRI related changes of sexual functioning have been reported after single doses concerning premature ejaculation [START_REF] Patel | Central regulation of ejaculation and the therapeutic role of serotonergic agents in premature ejaculation[END_REF] as well as after 8 days of intake concerning a broader spectrum of sexual functions including libido and sexual arousal [START_REF] Dunn | Assessing the onset of antidepressant-induced sexual dysfunction using interactive voice response technology[END_REF].

On the seventh day, subjects were scheduled for an fMRI scan two hours after intake of the last capsule.

Task and stimuli: In the scanner, a standard block design with prolonged visual stimulation using erotic and neutral video clips was used. Erotic video clips showed sexual interactions between one man and one or two women (petting, oral sex and vaginal intercourse) extracted from commercial adult films. The neutral video clips depicted men and women in emotionally neutral, non-erotic interactions (at a shop, at the airport). Nine video clips of each type were presented at a length of 20 sec each, separated by a 20 sec interstimulus interval with a white fixation cross shown on a black screen. Video clips were presented in a pseudo-randomized order with a maximum of two consecutive clips of the same type. After preprocessing, first level analyses were performed for each subject. According to the general linear model we defined regressors to analyze each of the two types of video stimuli (erotic, non-erotic). Video blocks were modeled as timely extended events of 20 sec and convolved with the hemodynamic response function. The 6 realignment parameters modeling residual motion were also included in the individual models. The individual contrast images for the erotic and non-erotic condition were then included in a second level group analysis using an ANOVA model with condition (EROTIC/NON-EROTIC) as the first factor. Treatment (PLAC/BUP/PAR) was added as a second factor with three levels to test on significant interaction effects of medication on the contrast of erotic vs. non-erotic stimuli. Inference of significant treatment effects was computed using directed condition-by-treatment t-contrasts for pairwise differences between treatment levels. To account for the post-hoc character of these tests, their computation was inclusively masked by an omnibus F-test on condition-by-treatment interactions within the 2x3 ANOVA. The statistical threshold for this mask was set to a value of p<0.005 uncorrected to avoid false negative results due to overly strict thresholding. However, significant treatment effects were inferred only if results of the post-hoc t-contrasts survived a statistical threshold of p<0.05 false discovery rate (FDR) corrected to account for multiple comparisons, and if clusters of significant voxels comprised at least 10 contiguously significant voxels.

In a second step, a conservative omnibus F-test was applied thresholded at p<0.05 with false discovery rate (FDR) corrections for multiple comparisons. [START_REF] Lingjaerde | The UKU side effect rating scale. A new comprehensive rating scale for psychotropic drugs and a cross-sectional study of side effects in neuroleptic-treated patients[END_REF]) by one of the study physicians (BA or AS) on each visit. Repeated-measures ANOVAs and post-hoc paired t-tests were applied to analyse reaction time and questionnaire results.

RESULTS

Questionnaires and neuropsychological tasks

Results from the ADS scale (mean: 6.9, SD 5.0) confirmed that none of the subjects suffered from depression at the time of the study. 56% of the subjects complained about side effects of any kind under placebo according to the UKU side effects scale. There were complaints over a wide range of different symptoms at a rate of 67% under bupropion and of 83% under paroxetine, without reaching significance when compared to placebo (ANOVA: F(2,34)=2.58, p=0.09). Particularly, subjective ratings of sedation did not differ (one-way ANOVA: F(2,34)=2.06, p=0.14) between treatments.

Upon enrolment subjectively rated sexual function as assessed by the mean cumulative score of the MGH-SFQ was 11.06 (SD 1.7). Above normal values (onesample t-test, p < 0.05) of more than 2 points per subscale were observed only for question (5) concerning sexual satisfaction (mean = 2.67, SD = 1.0), indicating minimal sexual dissatisfaction. However, subjective MGH-SFQ ratings differed significantly between medications (repeated measurements ANOVA: F(2,34)=5.05, p=0.01) with average overall scores of 12.0 (SD 2.4) under placebo, 11.8 (SD 2.9) under bupropion and 14.6 (SD 4.3) under paroxetine (figure 2A). Post-hoc paired ttests confirmed significantly higher overall scores under paroxetine as compared to placebo (t(17)=2.58, p=0.02) or bupropion (t(17)=2.30, p=0.03). No significant differences were observed when bupropion was compared against placebo.

Analyzing rating scores of each of the 5 MGH-SFQ subscales revealed a significant interaction effect of factors treatment and scale (repeated measurements ANOVA: F(8,136)=2.41, p=0.02) with increases of subjective ratings (paired t-tests, p < 0.05) concerning diminished sexual arousal and diminished ability to achieve orgasm under paroxetine as compared to both, enrolment and placebo ratings (figure 2B). It is of note, that the ability to get an erection was not affected by any of the treatments. Furthermore, none of the symptoms caused marked distress or interpersonal difficulty or otherwise justified a clinical diagnosis of sexual dysfunction.

Mean reaction times in the neuropsychological tests after scanning did not differ between treatments for the tests RT1 and RT2 (simple reaction without/with warning tone, one-way ANOVAs: RT1: F(2,34)=0.11, p=0.89; RT2: F(2,34)=0.65, p=0.53). For RT3, divided attention, we found a significant effect of medication (F(2,34=4.40, p=0.02)). Mean reaction times under placebo (mean 601.7 ms, SD 56.7) were significantly faster than under both drugs, bupropion (mean 629.3 ms, SD 52.0; t(17)=2.46, p=0.02) and paroxetine (mean 642.7ms, SD 69.8, t(17)=3.11, p=0.006).

No significant difference was found for bupropion vs. paroxetine (t(17)=0.78, p=0.45).

fMRI results

Main effect of erotic stimulation under placebo: Contrasting erotic minus non-erotic visual stimulation under placebo revealed significantly (p<0.05 FDR corrected) increased activations along the anterior cingulate cortex (ACC), in the amydgala, hypothalamus, ventral striatum, midbrain, lateral occipital cortex, precuneus and the fusiform gyrus.

Treatment effects: Significant results (p < 0.05, FDR corrected) of the directed posthoc tests on pairwise differences between treatment levels are summarized in table 1. Compared to placebo, decreased activation under paroxetine was significant in midbrain structures, the ventral striatum as well as in three different sections along the cingulate gyrus (sgACC, pgACC and aMCC). Comparing bupropion against placebo, only aMCC activation was decreased while the pMCC showed increased activity. Contrasting bupropion against paroxetine, increased activation was evident in the sub-and pregenual ACC as well as in the pMCC. Differential signals along the cingulate gyrus under the three different treatments are depicted in figure 3.

Furthermore, bupropion was associated with increased activation compared to both, paroxetine and placebo, in various brain regions including the SLEA, ventrolateral and dorsomedian thalamus, parahippocampus, lateral orbitofrontal cortex and fusiform gyrus (table 1).

To test on the reliability of the inclusive mask (p < 0.005, uncorrected) used for the post-hoc testing above, the same omnibus F-test on treatment effects used to create the mask was replicated at a statistical threshold of p < 0.05, FDR corrected. This test revealed again significant effects in the midbrain, the sub-and pregenual ACC (sgACC and pgACC), the anterior and posterior mid-cingulate cortex (aMCC and pMCC), the left sublenticular extended amygdala (SLEA), the left ventral striatum, and the left parahippocampal area (see also table 1).

To further investigate drug effects on the fMRI signal, signal time courses were extracted from three prototypical regions (figure 4): 1) the midbrain which, like rostral ACC regions, showed decreased activations under paroxetine as compared to placebo and bupropion, 2) the extended amygdala as a representative brain region for the network with increased activation under bupropion 3) the aMCC as the only brain region with decreased activation under both drugs compared to placebo. Paired t-tests of differences in signal time courses at single time points indicated prolonged differential effects in midbrain and particularly the extended amygdala under bupropion as compared to placebo. Differential effects in all three brain regions were most pronounced in the first part of the video stimulation.

Correlation analyses: Whole brain analyses did not reveal any significant correlations between functional activations and subjective ratings, reaction time data or medication blood levels (even when tested at different, rather liberal thresholds).

However, a significant correlation (figure 3) was observed between total MGH-SFQ score and averaged parameter estimates obtained from the pMCC region (voxels significant in the omnibus F-test at p<0.05 corrected). This region had been selected for further investigation based on previous literature showing correlations between brain activation in the mid-cingulate cortex with behavioural measures of sexual functioning [START_REF] Redoute | Brain processing of visual sexual stimuli in human males[END_REF][START_REF] Moulier | Neuroanatomical correlates of penile erection evoked by photographic stimuli in human males[END_REF][START_REF] Georgiadis | Dynamic subcortical blood flow during male sexual activity with ecological validity: a perfusion fMRI study[END_REF]. The more the brain activation decreased under paroxetine compared to placebo, the more overall ratings of sexual dysfunction (paroxetine minus placebo) increased (r=0.42; p=0.041). Furthermore, individual blood levels of paroxetine were also significantly (r=0.40, p=0.050) correlated with the differential neural activation (figure 5). No further significant correlations with rating scores, blood levels, or reaction times were observed in other cingulate areas significant in the above interaction analysis (particularly, not the aMCC). Computation of meaningful correlations between brain activations and behavioural ratings under bupropion was not possible due to the small variance of the rating scores with zero differences of ratings (placebo minus bupropion) in half of the subjects.

DISCUSSION

We investigated the effects of one week administration of the SSRI paroxetine and the dopamine and norepinephrine reuptake inhibitor bupropion in a double-blind placebo-controlled cross-over design in healthy male volunteers. We observed increased subjective ratings of sexual dysfunction under paroxetine compared to placebo and bupropion. Specifically, subjective sexual arousal and the ability to achieve orgasm were significantly impaired under paroxetine while the ability to get an erection, ratings of sexual interest and sexual satisfaction were not significantly altered under this drug.

Behavioural effects were accompanied by specific functional alterations in brain regions previously associated with sexual function in humans. Brain activation related to dynamic visual erotic stimulation under medication revealed antipodal results in two different networks. Compared against placebo and bupropion, paroxetine attenuated brain activation in neural structures comprising the midbrain, the nucleus accumbens, and sub-and pregenual anterior cingulate cortex. By contrast, bupropion led to an enhanced and prolonged activation in the parahippocampus, the amygdala (SLEA), thalamus and fusiform gyrus when compared against placebo or paroxetine.

Bupropion and paroxetine were associated with reduced activation within the anterior mid-cingulate cortex (MCC) and activation of the posterior MCC upon erotic stimulation was increased under bupropion. Individual parameters of decreased neural activation in the pMCC under paroxetine correlated with individually increased overall ratings of sexual dysfunction and with individual drug concentrations obtained from plasma samples.

Blunted anterior cingulate activation under Paroxetine

According to the four-region neurobiological model of human cingulate cortex architecture [START_REF] Palomero-Gallagher | Receptor architecture of human cingulate cortex: evaluation of the four-region neurobiological model[END_REF][START_REF] Vogt | Structural and functional dichotomy of human midcingulate cortex[END_REF], the four cingulate foci with differential medication effects are related to areas along the cingulate gyrus with distinct cytoarchitectonic and functional properties. This model postulates that subgenual ACC (sgACC) and pregenual ACC (pgACC) are reciprocally connected with the amygdala and are involved in emotional and autonomic processing. The aMCC is associated with reward processing and is active during fear. The pMCC has been related to error detection and is not primarily activated by simple emotions [START_REF] Phan | Functional neuroanatomy of emotion: a meta-analysis of emotion activation studies in PET and fMRI[END_REF][START_REF] Vogt | Structural and functional dichotomy of human midcingulate cortex[END_REF]. Paroxetine led to decreased fMRI activations in all of these regions along the cingulate gyrus. Particularly sgACC with its high density of 5-HT1A receptors in Brodman area 25 being the primary target of SSRI treatment [START_REF] Haddjeri | Long-term antidepressant treatments result in a tonic activation of forebrain 5-HT1A receptors[END_REF], has been postulated to be critical for understanding effects of antidepressant therapy [START_REF] Ressler | Targeting abnormal neural circuits in mood and anxiety disorders: from the laboratory to the clinic[END_REF]. Decreased sgACC activation under paroxetine is in line with findings of treatment effects in depression [START_REF] Mayberg | Deep brain stimulation for treatment-resistant depression[END_REF], and with findings of another fMRI study investigating the effects of subchronic SSRI administration in healthy subjects using emotional faces [START_REF] Arce | Escitalopram effects on insula and amygdala BOLD activation during emotional processing[END_REF]. This may point to a receptor specific but functionally more general SSRI effect independent of the type of stimulus material.

Like the sgACC, also the more rostral pgACC has been found to be modulated by subchronic escitalopram administration upon aversive stimuli [START_REF] Simmons | Subchronic SSRI administration reduces insula response during affective anticipation in healthy volunteers[END_REF].

Activity of the pgACC has also been postulated to correlate with subjective sexual intensity and its hedonic value (Walter, et al 2008a) and was identified as a brain region specifically coding the interaction of sexual intensity and emotional value of a stimulus. In Redoute's model of sexual arousal, activation of rostral ACC, comprising subgenual and pregenual parts, has been ascribed to the autonomic component of sexual behaviour mediating physiological readiness [START_REF] Redoute | Brain processing of visual sexual stimuli in human males[END_REF]. It is of note though, that sgACC and pgACC have also been characterized as brain regions coding emotional contents in a wider context [START_REF] Beckmann | Connectivity-based parcellation of human cingulate cortex and its relation to functional specialization[END_REF], of which autonomic reactions and self-referential processes may represent certain subsets. This suggests that paroxetine may lead to an impaired responsiveness of brain regions previously ascribed to the processing of emotional and autonomic functions that are associated with but also reach beyond the pure autonomic triggering of sexual arousal. Our observation that activation of the hypothalamus as a key autonomic component of sexual arousal [START_REF] Arnow | Brain activation and sexual arousal in healthy, heterosexual males[END_REF][START_REF] Ferretti | Dynamics of male sexual arousal: distinct components of brain activation revealed by fMRI[END_REF] did not show an effect of medication at the predefined statistical thresholds might be in line with this claim, although interpretation of this null result should be treated with caution.

A rather more specific role in the mediation of sexual dysfunction can be attributed to the mid-cingulate cortex. In a PET study using erotic stimulation, activation of this brain region was found to correlate with penile tumescence [START_REF] Redoute | Brain processing of visual sexual stimuli in human males[END_REF], and a recent study using fMRI could replicate this finding by demonstrating that the activation of the MCC correlated with penile tumescence in the early phase of sexual arousal and preceded physiological reactions [START_REF] Moulier | Neuroanatomical correlates of penile erection evoked by photographic stimuli in human males[END_REF]. [START_REF] Georgiadis | Dynamic subcortical blood flow during male sexual activity with ecological validity: a perfusion fMRI study[END_REF] reported blood flow changes in MCC and lateral hypothalamus that correlated with penis stimulation and perceived sexual arousal. In a recent study comparing effects of mirtazapine versus SSRIs (paroxetine and fluoxetine) during fMRI of dynamic erotic stimulation, patients with major depression showed decreased activation under SSRIs [START_REF] Kim | Treatment with Selective Serotonin Reuptake Inhibitors and Mirtazapine Results in Diefferential Brain Activation by Visual Erotic Stimuli in Patients with Major Depressive Disorder[END_REF] in cingulate areas in close proximity to the junction of the pMCC and posterior cingulate cortex. This pattern of results together with the present observation of decreased pMCC activation under paroxetine correlating with increased subjective sexual dysfunction supports a rather specific role of the MCC in mediating sexual functioning which is affected by SSRIs.

SSRI-related alteration of midbrain and ventral striatum activation

SSRI-dependent modulations of activity in midbrain and ventral striatum align with recent evidence indicating that the functioning of these brain regions is tuned by serotonin [START_REF] Kranz | Reward and the serotonergic system[END_REF]. The positive activation of these regions upon erotic stimulation in the sense of a primary reward under placebo adds to the accepted interpretation of these brain regions as part of the dopaminergic reward system [START_REF] Kranz | Reward and the serotonergic system[END_REF]. Despite of the inconsistencies concerning the general role of serotonin in reward processing with reports on reward increasing and on reward decreasing effects of serotonin, the latter results seem more prevalent. Particularly, diminished sexual desire and pleasure under SSRIs have been associated with the inhibition of dopaminergic reward circuits [START_REF] Kranz | Reward and the serotonergic system[END_REF]. This inhibition of dopamine transmission by SSRIs has been shown to be mediated by 5-HT2 receptors [START_REF] Alex | Pharamcologic mechanisms of serotonergic regulation of dopamine neurotransmission[END_REF], and research in animals demonstrated inhibitory effects of serotonin in the lateral hypothalamus upon sexual behaviour [START_REF] Lorrain | Extracellular serotonin in the lateral hypothalamic area is increased during the postejaculatory interval an d impairs copulation in male rats[END_REF] and on dopamine release in the ventral striatum [START_REF] Lorrain | Lateral hypothalamic serotonin inhibits nucleus accumbens dopamine: implications for sexual satiety[END_REF]. A 5-HT2A receptor polymorphism in humans has been reported to modulate sexual arousal under SSRIs in patients with depression [START_REF] Bishop | Serotonin 2A -depression and SSRI-associated sexual side-effects[END_REF]. SSRIs have also been reported to be less effective in reducing reward-related symptoms of depression like loss of interest and motivation [START_REF] Price | Emotional side-effects of selective serotonin reuptake inhibitors: Qualitative study[END_REF]. By contrast, greater positive effects in this respect have been ascribed to antidepressants with enhancing effects on catecholamine levels [START_REF] Nutt | The other face of depression, reduced positive affect: the role of catecholamines in causation and cure[END_REF] like bupropion. Accordingly, paroxetine attenuated activation in brain regions that are associated with the rewarding, motivational aspects of sexual behaviour.

Activity increases of subcortical regions under bupropion

Consistent with the proposed opponency of dopamine and serotonin [START_REF] Daw | Oponent interactions between serotonin and dopamine[END_REF], effects under bupropion as a dopamine and norepinephrine reuptake inhibitor were almost contrary to the SSRI paroxetine in midbrain and ventral striatal areas.

Furthermore, differential effects under bupropion were found over a prolonged time course during video stimulation even when compared to placebo. Modulation of ventral striatal activation by the subjectively perceived sexual intensity of a stimulus has been described before (Walter, et al 2008a), and changes in activity of the ventral striatum have been reported in various contexts of a rewarding sexual stimulation [START_REF] Hamann | Men and women differ in amygdala response to visual sexual stimuli[END_REF][START_REF] Stark | Erotic and disgust-inducing pictures--differences in the hemodynamic responses of the brain[END_REF]. Dopamine agonists have been shown to induce penile erection in animals and humans [START_REF] Carson | Central nervous system-acting agents and the treatment of erectile and sexual dysfunction[END_REF]) and have been suggested as promising agents for the treatment of sexual dysfunction (Albersen, et al). Dopamine agonists have further been related to hypersexuality in the treatment of parkinson's disease (for review see [START_REF] Voon | Chronic dopaminergic stimulation in Parkinson's disease: from dyskinesias to impulse control disorders[END_REF]). Impaired recruitment of the midbrain was suggested as a correlate of decreased sexual interest in an fMRI study on pedophilia [START_REF] Walter | Pedophilia is Linked to Reduced Activation in Hypothalamus and Lateral Prefrontal Cortex During Visual Erotic Stimulation[END_REF]. These findings support the interpretation that increased activation of the ventral striatum and the midbrain as dopaminergic brain regions may represent a neural correlate of increased responsiveness to sexual stimuli triggered by the dopamine agonistic properties of bupropion. This effect may also explain the concomitantly increased activation of the thalamus, parahippocampus and SLEA, since these structures are heavily interconnected. According to animal studies, the mediodorsal thalamus has subcortical connections to the ventral tegmental area and the midbrain's periaqueductal grey, to the amygdala, the SLEA and the hypothalamus [START_REF] Erickson | Subcortical afferents to the lateral mediodorsal thalamus in cynomolgus monkeys[END_REF][START_REF] Ongur | The organization of networks within the orbital and medial prefrontal cortex of rats, monkeys and humans[END_REF]. The thalamus and amygdala have already been related to the emotional dimension of sexual arousal [START_REF] Ferretti | Dynamics of male sexual arousal: distinct components of brain activation revealed by fMRI[END_REF][START_REF] Redoute | Brain processing of visual sexual stimuli in human males[END_REF], and particularly the mediodorsal proportion of the thalamus was found active for both, erotic and emotional stimulation [START_REF] Walter | High resolution fMRI of subcortical regions during visual erotic stimulation at 7 T[END_REF]. Recently, a region around the SLEA with similar stereotactic coordinates as in the present study was reported to show the strongest blood flow changes when penile erection and perceived sexual arousal peaked and was suggested to potentially relate to orgasmic pleasure [START_REF] Georgiadis | Dynamic subcortical blood flow during male sexual activity with ecological validity: a perfusion fMRI study[END_REF]. Together with cortical regions like the MCC, posterior cingulate and anterior insula, the network of these subcortical regions has been referred to as the 'salience network', integrating functions of conflict monitoring, autonomic functions and reward-processing [START_REF] Seeley | Dissociable intrinsic connectivity networks for salience processing and executive control[END_REF]. Activation in pMCC was increased under bupropion as the cortical part of the network which has been suggested to represent the approach component of sexual arousal [START_REF] Redoute | Brain processing of visual sexual stimuli in human males[END_REF].

Unidirectional effects of both antidepressants

Despite opposite effects in extended networks, both drugs led to a decrease of activation compared to placebo in one subregion of the cingulate cortex, the aMCC.

Since both drugs otherwise led to differential effects upon processing erotic stimulation this unidirectional effect does not integrate in a stimulation-bound interpretation. Alternatively one might assume that the activation decrease in the aMCC may reflect an alteration of neural functioning not necessarily interfering with sexual functioning. This might be supported by the observation that both drugs also led to unidirectional behavioural effects with impaired performance on the divided attention task. This task puts the subject in a dual-task situation where task-set processes have been suggested to constitute a core resource of limited capacity shared across different tasks [START_REF] Dosenbach | A Core System for the Implementation of Task Sets[END_REF]. Midcingulate activation in the vicinity of the aMCC focus was suggested to be part of a frontoparietal network exerting active and adaptive online control helping to direct selective attention [START_REF] Dosenbach | Distinct brain networks for adaptive and stable task control in humans[END_REF]. Although we could not observe substantial across subjects correlations between reaction time and decreased activity of the aMCC, one might speculate that impaired divided attention and decreased aMCC activation may both result from an impaired task set processing under both drugs presumably via different receptor pathways. Interactions of dopamine receptors with NMDA receptors [START_REF] Chen | Interaction of dopamine D1 and NMDA receptors mediates acute clozapine potentiation of glutamate EPSPs in rat prefrontal cortex[END_REF] with their particularly high density in aMCC (but not other ACC subregions; Palomero-Gallagher, et al 2009) could help to explain the differential effects of bupropion on aMCC and pMCC.

Conclusion

Enhancement of serotonergic and noradrenergic/dopaminergic neurotransmission led to differential effects in the subjective experiences of sexual functioning accompanied by opposite drug effects on local neural activities upon erotic stimulation. Overall, most brain activations elicited by erotic movies were reduced under paroxetine but not under bupropion. Particularly, under paroxetine neural activities decreased in the ventral striatum, midbrain and distinct areas along the cingulate gyrus. These regions have already been described by others to mediate aspects of motivational and emotional processes related to sexual behaviour although their specific role awaits further research. The combined noradrenalin/dopamine reuptake inhibitor bupropion showed opposite fMRI effects and was associated with a prolonged and increased activation of brain regions previously related to the processing of rewarding and salient stimuli. If clinically possible, this suggests enhancement of dopaminergic transmission as a plausible add-on treatment to counteract the side effects of antidepressant SSRI medication on sexual functioning which impose a high risk on medication adherence and hence therapeutic success. The table shows results of post-hoc testing of pairwise differences between treatments at p<0.05 FDR corrected. Post-hoc testing was locally constrained to voxels surviving a threshold of p<0.005 in the 2x3 ANOVA with factors condition (erotic, non-erotic) and treatment. Brain areas with significant interaction effects in the 2x3 ANOVA even at the more conservative threshold of p < 0.05, FDR corrected are marked with (*). No significant interactions were observed for Paroxetine > Placebo or Paroxetine > Bupropion. #: activation is part of cluster above; Z: z-score; NV: number of contiguously significant voxels; peak coordinates of clusters are are MNI (Montreal Neurological Institue) normalized stereotactic coordinates: -x: left from the anterior commissure (AC); -y: posterior from AC; -z: inferior from AC. ACC: anterior cingulate cortex; MCC: midcingulate cortex; FC: frontal cortex; BA: Brodman area

TITLES AND LEGENDS TO FIGURES

  fMRI acquisition: A 3.0 Tesla Magnetom ALLEGRA Scanner (Siemens, Erlangen, Germany) equipped with a head coil was used to acquire T1 anatomical volume images (1x1x1 mm voxels) and functional magnetic resonance images. 23 transversal slices were acquired with an image size of 64×64 pixels and a field of view of 192 mm. Slice thickness was 3 mm with 0.75 mm gap resulting in a voxel size of 3×3×3.75 mm. Images were centered on basal structures of the brain including subcortical regions of interest (basal ganglia and prefrontal regions). Functional images were recorded using T2*-sensitive gradient echo echo-planar imaging measuring changes in BOLD-contrast. 487 volumes were obtained during viewing of video clips at a TR of 1500 ms (TE 35 ms, flip angle 90°). fMRI analysis: Image preprocessing and statistical analyses were carried out using Statistical Parametric Mapping (SPM5, Wellcome Trust Centre for Neuroimaging, London, UK) with a random effects model for group analyses. Preprocessing of the individual functional scans included realignment to correct for motion artifacts, slice timing, spatial normalization to a standard template (Montreal Neurological Institute, MNI) and smoothing with an 8 mm full width at half maximum (FWHM) Gaussian kernel. Intrinsic autocorrelations were accounted for by AR(1) and low frequency drifts were removed via high pass filtering.

  Neuropsychological testing and additional questionnaires: After scanning and blood sampling, subjects performed three simple reaction time tasks (subtests from the Testbattery for Attentional Performance, Zimmermann & Fimm, Psychologische Testsysteme, Herzogenrath, Germany): simple reaction to a visual stimulus (RT1), reaction to a visual stimulus after a warning tone (RT2), and divided attention (RT3), involving reaction to distinct visual and auditory stimuli at the same time. Reaction time tasks were applied to assess for general medication effects on attention and concentration as potential moderators of treatment effects. Subjects also completed the self-report questionnaires on side effects and sexual functioning (MGH-SFQ, for detailed description see supplements) during the past week on each occasion. Cumulative ratings on the MGH-SFQ ranged from 5 (minimal value: improvement of sexual functioning) over 10 (sexual functioning unchanged compared to normal) to 30 (maximal value: sexual functioning markedly impaired compared to normal). Ratings above 10 indicate subjectively impaired sexual functioning as compared to normal in the sense of subjective sexual dysfunction. Subjective side effects were additionally assessed in a medical interview with open questions and a structured part (UKU side effects scale,

Figure 1 :

 1 Figure 1: Study design. 6 subjects were assigned to each of the three randomization

Figure 2 :

 2 Figure 2: (A) Group averaged cumulative rating scores obtained from the 5-item

Figure 3 :

 3 Figure 3: Significant treatment effects (p<0.05 FDR corrected) along the anterior

Figure 4 :

 4 Figure 4: Statistical parametric maps (SPMs) and fMRI signal time courses extracted

Figure 5 :

 5 Figure 5: Significant (p<0.05) correlations of fMRI activation in the pMCC region of

Table 1 :

 1 Significant condition-by-treatment interaction effects

		Placebo > Paroxetine		Placebo > Bupropion		Bupropion > Placebo	Bupropion > Paroxetine
	Video:	Z	NV	Peak Coord.	Z	NV	Peak Coord.	Z	NV	Peak Coord.	Z	NV	Peak Coord.
	erotic vs. non-erotic stimuli
				x/y/z			x/y/z			x/y/z			x/y/z
	Cingulate Cortex
	Subgenual ACC*	4.21	46	-12/32/-6							3.61	37	-14/32/-6
	Pregenual ACC*	4.08	104	14/46/4							2.95	94	12/44/6
	Anterior MCC*	3.37	30	14/32/32	4.67	90	16/34/30
	Posterior MCC*							3.30	102	16/20/34	4.43	119	16/24/36
	Subcortical structures
	Midbrain*	4.72	150	-4/-24/-14							5.29	485	-4/-24/-14
	Ventral striatum, right	3.04	16	6/14/-6							3.66	30	10/12/-6
	Ventral striatum, left										4.24	650	-18/12/-6
	Extended amygdala, left*							4.82	650	-22/0/-10	4.20	#	-22/0/-10
	Extended amygdala, right							3.29	125	26/-8/-12	4.16	137	26/-6/-10
	Ventrolateral Thalamus, right							3.64	44	18/-14/6	3.22	44	12/-14/2
	Ventrolateral Thalamus, left*							3.80	285	-22/-14/14	4.33	299	-22/-14/14
	Dorsomedian Thalamus							2.43	#	-6/-12/8	3.74	#	-4/-10/6
	Cortical activations
	Lateral orbital FC* (BA11/47)							3.64	58	-36/40/-8	4.04	61	-34/36/-6
	Medial FC, BA 6							3.70	140	52/0/30	3.60	129	56/6/34
	Medial/superior temporal							3.79	268	38/-56/30	4.55	271	36/-58/28
	Gyrus* (BA 39/40)
	Fusiform gyrus, left (BA 19/37)							3.50	44	-34/-62/-4	3.79	44	-36/-62/-6
	Fusiform gyrus, right* (BA 19/37)										3.76	56	38/-40/-12
	Parahippocampus, left*							3.58	450	-22/-34/-6	4.38	450	-20/-34/-6
	Parahippocampus, right							4.00	142	42/-20/-14	2.85	61	40/-22/-10
	Parahippocampus, right							3.99	56	36/-38/-12
	Cerebellum, left*							3.85	524	-42/-56/-24	4.05	521	-42/-56/-24
	Cerebellum*							3.77	#	2/-74/-28	4.05	#	-8/-70/-18
	Cerebellum, right							3.35	22	22/-48/-18	3.00	22	18/-50/-18

ACKNOWLEDGMENTS

We thank Prof. Dr. C. Hiemke and his staff at the University of Mainz (Germany), Department of Psychiatry and Psychotherapy, for measurements of paroxetine and bupropion blood serum levels.

DISCLOSURE/CONFLICT OF INTEREST

All authors declare that they have received no financial support or compensation from any individual or corporate entity over the past 3 years for research or professional service, except for income received from their primary employers, and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.


M Putzeist

¹ A K Mantel-Teeuwisse ¹ ²

C C Gispen-De Wied²

A W Hoes²

H G Leufkens¹

A K Mantel-Teeuwisse

Regulatory Scientific Advice in drug development: Does company size make a difference?

Keywords: Drug development, Scientific Advice, Company size

Purpose To assess whether the content of Scientific Advice (SA) questions is associated with company size. This may help to learn more about the knowledge, strategic and regulatory gaps companies face during drug development.

Methodology

A cross-sectional analysis was performed of SA provided by the Dutch Medicines Evaluation Board (MEB) in 2006-2008. Definition of company size was based on ranking by total revenues (Scrip"s Pharmaceutical Company League Tables 2008). The content of each SA question was scored according to predefined domains (Quality, Nonclinical, Clinical, Regulatory and Product Information), their subdomains e.g. efficacy, and a selection of additional content variables e.g. endpoints, choice of active comparator.

Results

In total 201 SA documents, including 1,087 questions could be identified. Small, medium-sized and large companies asked SA 110 (54.7%), 40 (19.9%) and 51 (25.4%) times, respectively. Clinical questions were asked most often (65.9%), mainly including efficacy (33.2%) and safety questions (24.0%). The most frequently asked topics were overall efficacy and safety strategy.

Small companies asked quality and non-clinical questions more often (p<0.001) and clinical questions less frequently than large companies (p=0.004). Small companies asked significantly more clinical questions about pharmacokinetics, including bio-equivalence, than medium-sized and large companies (p<0.001).

Conclusion

The array of topics addressed in SA provides an interesting outlook on what industry beliefs is still unresolved in drug development and is worthwhile to discuss with regulators.

Company size is associated with the content of scientific advice questions. MEB advice accommodates both innovative and non-innovative drug development.

Introduction

Marketing authorization of a new medicinal product is a critical step in the access to innovative therapies that are needed to fill current pharmaceutical gaps and unmet medical needs. Despite the increasing number of applications for marketing authorization in Europe, the proportion of applications with a negative decision remains relatively high, around 25%-30%, and was even 40% for New Active Substances with an approval outcome in 2009. (

1)

(2) There is increasing concern about the obvious gap between the output of drug development and registration strategies applied by companies, and EU-regulatory expectations. (3) (4) Industry response to this development echoes that improved communication with regulatory authorities during drug development is needed. [START_REF] Emea | Innovative Drug Development Approaches; Final report from the EMEA-CHMP Think Thank Group on Innovative Drug Development[END_REF] Additional regulatory requirements in recent years have complicated the authorization procedure and have made innovative drug development more costly. Besides, with complex biologicals, advanced therapies and personalized medicines becoming more important, the need for more specific guidance in drug development has increased. [START_REF] Emea | The European Medicines Agency Road map to[END_REF] [START_REF] Ema | The European Medicines Agency Road Map to[END_REF] Before and during the marketing authorization procedure of a medicinal product, pharmaceutical companies have various opportunities to discuss critical issues in the drug development process with regulators. A continuous and ongoing regulatory dialogue between pharmaceutical industry and regulatory authorities has often been recommended as a strategy to support innovative drug development in an efficient and tailored way. [START_REF] Emea | Innovative Drug Development Approaches; Final report from the EMEA-CHMP Think Thank Group on Innovative Drug Development[END_REF] [START_REF] Ormarsdottir | European regulatory perspectives for innovative therapies[END_REF] (3) (9) (10) A relevant part of scientific regulatory dialogue is so-called Scientific Advice (SA), the opportunity of (early) communication between a company and a regulatory authority on quality, non-clinical and various clinical aspects (e.g. study design, choice of endpoint, indication) of drug development. In Europe an increasing proportion of market application authorizations are preceded by SA; 47% of all applications in 2007 received SA and in 2008 this percentage was 56%. [START_REF] Emea | Survey on the performance of EMEA scientific procedures for medicinal products for human use[END_REF] An applicant for SA can be a pharmaceutical company or (a group of) scientist(s) who is developing a product. Applicants are encouraged to seek regulatory SA as many times as necessary, but both industry and authorities are not obliged to adhere to the advice received or committed to accept any result of a SA procedure. [START_REF] Emea | New Framework for SA & Protocol Assistance EMEA[END_REF] In Europe, SA can either be seeked at the European Medicines Agency (EMA) or at one or more of the national regulatory agencies. National regulatory agencies provide SA either as a response to national SA requests or as an answer to European SA requests, outsourced by the EMA Scientific Advice Working Party (SAWP) to one or two of its member countries according to expertise. A recent study looking at SA provided by EMA, demonstrated that the level of industry adherence to SA and company size were both predictors of a positive outcome in a marketing authorization procedure. The study also showed that among companies submitting a marketing application to EMA, large companies asked SA most frequently and were more adherent to the advice than medium-sized and small companies. [START_REF] Regnstrom | Factors associated with success of market authorisation applications for pharmaceutical drugs submitted to the European Medicines Agency[END_REF] Considering that adherence to SA is associated to a positive outcome in a marketing authorization procedure, and that variability in adherence to SA exists between companies, the question arises whether company size matters when looking at the type of SA that pharmaceutical companies are seeking. Answering this question may help to learn more about the knowledge, strategic and regulatory gaps companies face during drug development and how these differentiate between the various types of enterprises.

Methods

Study design and scientific advice characteristics

A cross-sectional analysis was performed of national SA provided by the Dutch Medicines Evaluation Board (MEB) in the years 2006-2008. SA documents were retrieved from the MEB SA Database. Requests for SA, which were rejected by the MEB for reasons of lack of expertise or previously received EMA advice, were excluded. In this study was looked at individual requests for SA, so follow-up SAs for a similar medicinal product were included.

Products for which SA was given in the study period were categorized according to anatomical main group of the ATC-classification. [START_REF]WHO Collaborating Centre for Drug Statistics Methodology[END_REF] In case an ATC-classification was missing, the anatomical main group was assessed based on the intended indication of the product. Secondly, products were categorized as new chemical substance (NCS; chemical substance not previously approved), generic (a product with identical qualitative and quantitative composition and similar pharmaceutical form as original product), biologicals (defined as vaccines, blood and blood components, allergenics, somatic cells, gene therapy, tissues and recombinant proteins) and new application of existing drugs (previously approved chemical substances for which a new indication was seeked, dosage form or other variation was being developed, in such a way that there was a need for additional efficacy and safety studies).

Company size was defined as small-, medium-sized and large, based on ranking by total revenue as reported in Scrip"s Pharmaceutical Company League Tables 2008. [START_REF] Markovaara | Scrip Pharmaceutical Company League tables[END_REF] Companies were defined large if ranked 1-20, medium-sized if ranked 21-150 and small if the company was not on the ranking list. This definition was in line with a previous study on SA. [START_REF] Regnstrom | Factors associated with success of market authorisation applications for pharmaceutical drugs submitted to the European Medicines Agency[END_REF] For each advice we evaluated whether previous advice for the same product had been requested at the MEB, whether parallel advice had been asked for at another national regulatory agency, or both.

Data collection: Characteristics of questions

Each SA consisted of a variable number of questions asked by companies. All questions from SA given by the MEB in 2006, 2007 and 2008 were collected and analyzed in a standardized fashion. Each question, being the unit of analysis, was scored separately according to variables at three different levels: domains, subdomains and content variables (Figure 1). At the first level the question content was analyzed according to the domains "Quality, Non-clinical, Clinical, Regulatory and Product Information". Scoring more than one domain was allowed, for example when a clinical and a product information issue were discussed in the same question.

Secondly, subdomains were formulated and scored for questions in the non-clinical and clinical domains. The subdomains of the non-clinical domain were "Pharmacodynamics", "Pharmacokinetics" and "Toxicology". For domain "Clinical" a subdivision was made for questions with regard to "Pharmacodynamics" (including Dose Finding studies), "Pharmacokinetics" (including bio-equivalence studies), "Efficacy" or "Safety". Again, scoring more than one subdomain was allowed.

Additionally, at the third and most detailed level each question was scored by a selection of content variables, e.g. primary endpoint, choice of active comparator, trial duration and overall efficacy programme. The content variables were selected based on general regulatory requirements of the drug development process and existing EMA regulatory guidelines. In this third step, a distinction was made between specific and strategic questions. Strategic questions were defined as questions in which general feedback was asked about e.g. the complete quality programme or the clinical efficacy programme. An example of a strategic question was: "Does the MEB agree that the results of the clinical efficacy programme will be sufficient to market approval of the product in the specific indication"? Specific questions were defined as being related to specific topics of the development plan in one single study. An example of a specific question is: "Does the MEB agree with the chosen primary endpoints for this indication?"

Data analysis

Associations between the type of SA questions and company size were assessed by Pearson"s Chi-square analysis. P-values were calculated for each variable. Differences in average number of questions were assessed by a one way ANOVA-test.

Results

During the study period the MEB provided 214 times SA. Thirteen advice documents were missing (4 and 9 documents in 2006 and 2007, respectively). In total 201 SA documents, including 1,087 questions could be identified. SA has been provided for 187 single products, whereas 117 single companies (80 small, 21 medium sized and 16 large companies) received SA in the study period. The general SA characteristics are given in Table 1.

SA was most frequently given for nervous system drugs (24.9%), but for a variety of other therapeutic areas SA was provided as well. More than 60% of SA was given for generics and new applications of existing drugs. Small, medium-sized and large companies asked SA 110 (54.7%), 40 (19.9%) and 51 (25.4%) times respectively. More than 40% of the companies seeking for SA had previously received advice for the same drug at the MEB or another national agency.

On average five questions per SA were asked (table 2). Clinical questions were asked most frequently, 716 times (65.9%). Within the clinical subdomain, efficacy and safety questions were most frequently asked, 361 (33.2%) and 261 (24.0%) times respectively.

Small companies asked significantly lower numbers of questions per SA compared to medium and large sized companies (p<0.001). Large and medium sized companies asked significantly more SA questions about new chemical entities than small companies (p<0.001). Small companies asked 70% of SA questions about drug development of generics and new applications of existing drugs. These small companies were a diverse representation of companies; generic companies (20%), innovative pharmaceutical or biotech companies (40%) and other companies mainly consisting of medical technology companies, working on new applications of drugs and consultants. Medium-sized companies, about 85% of these were innovative pharmaceutical or biotechnology companies, most frequently asked SA questions related to the development of biologicals (p< 0.001).

With regard to domain, the majority of questions asked by companies were about clinical development issues, while small companies were more visible on quality and non-clinical questions. One out of five SA questions was on regulatory issues, with no difference between types of companies. Within the clinical domain, small companies asked significantly more often about pharmacokinetics, including bio-equivalence, than medium-sized and large companies. These companies less often posed efficacy questions than large companies (24.4% vs. 44.8% p<0.001). The proportion of safety and strategic questions was not associated with company size.

In Figure 2 the overall top ten of most frequently asked topics on the most detailed third level of variables is given, showing a strong preference for clinical topics. Overall, most frequently asked questions were questions about overall efficacy strategy (9.6%) and safety strategy (9.1%). Besides, strategy questions about the clinical pharmacokinetic programme were in the top ten. Indication, primary endpoints, dosing and study population were examples of popular specific topics. More details of the ten most frequently asked topics are given in Table 3.

Discussion

One of the main findings in this study was that indeed SA, provided by a national authority is different, both quantitatively and in terms of kind of questions, when looking at company size. Our content analysis of SA demonstrates that the majority of questions raised by companies, particularly the large ones, were about clinical drug development. Small companies spent more attention to quality and nonclinical issues than large companies.

The array of topics addressed in SA provides an interesting outlook, given all its limitations caused by strategic behaviour of companies and selective acceptance of SA by regulators, on what industry beliefs is still unresolved in drug development and is worthwhile to discuss with regulators.

Regnstrom et al. emphasized the importance of adherence to SA for a successful marketing approval. [START_REF] Regnstrom | Factors associated with success of market authorisation applications for pharmaceutical drugs submitted to the European Medicines Agency[END_REF] The question arises whether our findings of companies" priorities in drug development are in line with the most often occurring major objections or factors for approval failure. A 2002 study with EMA data found that major objections raised by regulators in the marketing authorization procedure were lack of adequate randomized controlled trials to prove clinical efficacy and the occurrence of unresolved safety issues. (3) The EMA reported in 2008 that critical issues related to study design (39%), patient population (35%), endpoint (35%) and the magnitude of an effect (48%) were important drivers of a negative application.

(1) In a 2010 study with FDA data on orphan drugs Heemstra et al. found that failing to achieve primary endpoints and lack of description of appropriate target population were related to non-approval. [START_REF] Heemstra | Characteristics of orphan drug applications that fail to achieve marketing approval in the U.S[END_REF] Our study also showed that topics like study design, endpoints, study population and special safety issues were all in the top ten of most frequently addressed issues in SA.

Quality documentation is a particular bottleneck for Small and Medium sized Enterprises (SMEs). The EMA Small and Medium sized Enterprise office reported that quality documentation caused 41% of the major objections in application procedures of SMEs in 2008. [START_REF] Emea | [END_REF] Our results demonstrated that SMEs asked significantly more often about quality issues than large companies, the latter hardly discussed any quality issues. This implies that SMEs lack knowledge with regard to quality documentation or capacity to comply with the requirements. Some limitations of this study need to be discussed. Firstly, the scoring method of the SA, although highly standardized, may be susceptible to some subjectivity. In order to minimize this, we scored the questions according to strict definitions of content variables. These variables were derived from scientific regulatory documents and guidelines.

Secondly, our definition of company size differs from the official EU definition of SMEs.

According to the official SME definition, only 15 of 201 SA requests would have been classified as an SME request. This would create a group of "large companies" which was too heterogeneous to draw any conclusions about. Therefore, we based the SME definition on ranking by total revenue as reported in Scrip"s Pharmaceutical Company League Tables 2008, which was in line with a previous study on SA with EMA data. [START_REF] Regnstrom | Factors associated with success of market authorisation applications for pharmaceutical drugs submitted to the European Medicines Agency[END_REF] Another limitation of our study is that we did not investigate company"s reasons for asking SA. These reasons may range from a real interest in the answers to the questions raised in SA to expected positive effects on the regulatory process (and outcome) by the applicants from dialogue and alignment with regulators in general.

It should be noted that company budgets may drive the decision to ask SA. However, during a significant part of the research period the MEB provided SA free of charge. In addition, the costs that were introduced later are very limited, varying from 3500 to 8000 euro per advice.

Therefore we believe it is unlikely that the costs of SA have influenced our results and we do not consider this a limitation of our study.

The fact that some SA requests at the MEB were rejected may raise the question whether results of this study are representative for national SA provided by other regulatory agencies.

Advice requests were rejected when former advice was obtained from EMA, in case the complexity was expected to be better dealt with at EMA-level or when the indication of the product was outside the scope of the expertise of the MEB. Therefore the array of clinical areas represented in this study is also a reflection of national MEB expertise. Despite national expertise in e.g. central nervous system and cardiovascular products, the MEB gave SA about drug development in a broad range of therapeutic areas (Table 1). Similar broad ranges of therapeutic areas are expected for SA at other national regulatory agencies in the EU as well. In addition, when comparing the Dutch national SA with other national SA, the top ten of most frequently asked topics will probably not be influenced by differences in expertise because it consists of topics that are related to drug development in general. Therefore, we think our results give a well-balanced overview of issues in drug development.

Regulatory dialogue about challenging issues at the critical edge of drug development is seen as a key success factor for bringing new medicinal products with a positive benefit-risk to the patient. An EMA brainstorm session held with regulators and pharmaceutical industry representatives in 2007 made clear that a special need exists for dialogue about new high risk advanced therapies and technologies and for new scientific approaches in targeted drug development, such as validation of biomarkers, choice of study endpoints or better methods to identify treatment responders. Besides, the use of more flexible and adaptive study designs was raised as a key issue to be discussed in a dialogue with regulators. [START_REF] Emea | Innovative Drug Development Approaches; Final report from the EMEA-CHMP Think Thank Group on Innovative Drug Development[END_REF] According to the EMA, in particular many SMEs are active in the development of the highly innovative Advanced Therapy Medicinal Products (ATMPs). ( 14) SA related to such high risk advanced therapies and technologies are channeled to the EMA. In contrast, other small companies asked MEB-advice most frequently about generic applications, bio-equivalence and new application of existing drugs. One may argue that answers to these SA questions could also be found in regulatory guidelines. The need for such advice may be partly attributable to lack of experience in drug development or lack of clarity in existing guidelines.

The role of scientific advice also has bearing on the way companies formulate their questions. For all types of companies about 20% of all questions asked, were "strategic".

Further research should assess whether companies benefit more from asking specific or strategic questions. Besides, in further research national SA could be compared to European SA to assess whether strategic questions are asked on both levels and to evaluate commonalities and differences in the roles of European and national SA.

Moreover, the level of complexity of SA questions would give deeper insight information in the issues addressed. This would also enable further research on how complexity drives market authorization holders" behaviour when it comes to SA.

In conclusion, SA as provided by a regulatory authority provides a detailed outlook of unresolved issues in drug development. This picture is a function of industry presence in a certain country, of expertise at a national regulatory authority, but also of critical issues at the edge of regulatory decision making. Indeed, there is variability in how different companies deal with this. The results of this study show that company size is associated with the content of SA questions and that national SA accommodates both innovative and noninnovative drug development. Clinical pharmacology topics are at the top of issues discussed in SA, a finding that asks for more analysis on how fruitful industry, regulatory and academic clinical pharmacologists interact and align in order to stimulate drug innovation.

Figure 1 :

 1 Figure 1: Scoring method for SA questions

Table 1 Baseline characteristics of SA

 1

	Variables	Advice (N=201)
		(% of total no. of advice)
	Year
	2006	77 (38.3%)
	2007	47 (23.4%)
	2008	77 (38.3%)
	Total	201(100%)
	ATC-Code
	A/Alimentary tract & Metabolism	12 (6.0%)
	B/ Blood& Blood forming organs	16 (8.0%)
	C/ Cardiovascular system	34 (16.9%)
	G/ Genito-Urinary system & sex	15 (7.5%)
	hormones
	J/ Antiinfectives	13 (6.5%)
	L/ Antineoplastic &	29 (14.4%)
	Immunomodulating products
	N/ Nervous system	50 (24.9%)
	Other	32 (15.9%)
	Product Type EMEA
	NCE	43 (21.4%)
	Generic	59 (29.4%)
	New application of existing drug	64 (31.8%)
	Biological (including biosimilars)	34 (16.9%)
	Other (General advice)	1 (0.5%)
	Orphan drugs
	Orphan drug	3 (1.5%)
	Non-orphan drug	198 (98.5%)
	Company Size
	Small	110 (54.7%)
	Medium-sized	40 (19.9%)
	Large	51 (25.4%)
	Type of Registration Procedure
	Central	48 (23.9%)
	Decentral	42 (20.9%)
	Mutual Recognition Procedure	19 (9.5%)
	National	11 (5.5%)
	To be decided	75 (37.3%)
	Missing	6 (3.0%)
	Previous/ Parallel advice
	No previous	102 (50.7%)
	At MEB	17 (8.5%)
	At Other agencies	62 (30.8%)
	At MEB & other agencies	9 (4.5%)
	Missing	11 (5.5%)

Table 2 Company size relations with characteristics and content of SA questions

 2

	Questions N=1087	Small Pharma Medium	Large Pharma Total	P-values
			Pharma
	Total No. of Questions	431 (39.7%)	310 (28.5%)	346 (31.8%)	1087(100%) <0.001
	Average No. of Questions	3.9 (1-18)	7.8 (1-27)	6.8 (1-17)	5.4 (1-27)	<0.001
	SD	3.2	5.7	3.9	4.3
	ATC-Code
	A/Alimentary tract &	29 (6.7%)	0 (0.0%)	41 (11.8%)	70 (6.4%)	<0.001
	Metabolism
	B/ Blood& Blood forming	26 (6.0%)	66 (21.3%)	7 (2.0%)	99 (9.1%)	<0.001
	organs
	C/ Cardiovascular system	39 (9.0%)	63 (20.3%)	69 (19.9%)	171(15.7%)	<0.001
	G/ Genito-Urinary system &	6 (1.4%)	48 (15.5%)	60 (17.3%)	114(10.5%)	<0.001
	sex hormones
	J/ Antiinfectives	40 (9.3%)	15 (4.8%)	8 (2.3%)	63 (5.8%)	<0.001
	L/ Antineoplastic &	58 (13.5%)	30 (9.7%)	75 (21.7%)	163 (15.0%)	<0.001
	Immunomodulating products
	N/ Nervous system	114 (26.5%)	80 (25.9%)	58 (16.8%)	252 (23.2%)	0.003
	Other	119 (27.6%)	8 (2.6%)	28 (8.1%)	155 (14.2%)	<0.001
	Product Type EMEA
	NCE	47 (10.9%)	89 (28.7%)	173 (50.0%)	309 (28.4%)	<0.001
	Generic	125 (29.0%)	17 (5.5%)	31 (9.0%)	173 (15.9%)	<0.001
	New application of existing	178 (41.2%)	106 (34.2%)	101 (29.2%)	385 (35.4%)	0.002
	drug
	Biological (incl.biosimilars)	80 (18.6%)	98 (31.6%)	41 (11.8%)	219 (20.1%)	<0.001
	Other (General advice)	1 (0.2%)	0	0	1 (0.1%)	0.46
	Previous/parallel advice
	No previous	233 (54.1%)	90 (29.0%)	98 (28.3%)	421(38.7%)	<0.001
	At MEB	9 (2.1%)	38 (12.3%)	64 (18.5%)	111(10.2%)	<0.001
	At Other agencies	161 (37.4%)	154 (49.7%)	134 (38.7%)	449 (41.3%)	0.002
	At MEB & other agencies	6 (1.4%)	11 (3.5%)	32 (9.2%)	49 (4.5%)	<0.001
	Missing	22 (5.1%)	17 (5.5%)	18 (5.2%)	57 (5.2%)	0.973
	Domain
	Quality	43 (10.0%)	26 (8.4%)	5 (1.4%)	74 (6.8%)	<0.001
	Non-Clinical	69 (16.0%)	22 (7.1%)	19 (5.5%)	110 (10.1%)	<0.001
	Clinical	272 (63.1%)	192 (61.9%)	252 (72.8%)	716 (65.9%)	0.004
	Regulatory	86 (20.0%)	59 (19.0%)	60 (17.3%)	205 (18.9%)	0.649
	Product Information	11(2.6%)	22 (7.1%)	22 (6.4%)	55 (5.1%)	0.009
	Clinical Subdomain
	Pharmacodynamics (incl.	26 (6.0%)	13 (4.2%)	27 (7.8%)	66 (6.1%)	0.154
	Dose Finding)
	Pharmacokinetics (incl.BE)	123 (28.5%)	30 (9.7%)	41 (11.9%)	194(17.8%)	<0.001
	Efficacy	105 (24.4%)	101 (32.6%)	155 (44.8%)	361(33.2%)	<0.001
	Safety	97 (22.5%)	78 (25.2%)	86 (24.9%)	261(24.0%)	0.639
	Type of Questions
	Strategic Questions	93 (21.6%)	64 (20.6%)	65 (18.8%)	222 (20.4%) 0.627

Are there any specific aspects on safety you would like us to pay special attention to? Does the agency concur with the company's proposal to perform only ECGs in the proposed pivotal studies, given the absence of a QTc prolongation effect in a thorough QT study?

The recently initiated Phase II-III clinical trial has the following characteristics:"……" Is this trial design acceptable for definitive confirmation of the clinical benefit and of an acceptable safety profile of drug X?

Dosing

The appropriateness of the doses chosen for a clinical study.

The scheme for the individual dosing is a 10 mg/kg loading dose followed by a 5 mg/kg maintenance dose. The company considers increasing the maintenance dose if no adverse effects are seen.

Does the MEB agree to the proposed dosing regimen?

Study population

The appropriateness of the inclusion and exclusion criteria Does the Agency concur with the definition of the patient population to be studied in the Phase 3

Figure 2 Top ten Most frequently asked topics