Cortico-Amygdala-Striatal Activation by Modafinil/Flecainide Combination

Cortico-Amygdala-Striatal Activation by Modafinil/Flecainide Combination Background: Modafinil, a nonamphetaminic wake-promoting compound, is prescribed as first line therapy in narcolepsy, an invalidating disorder characterized by excessive daytime sleepiness and cataplexy. Although its mode of action remains incompletely known, recent studies indicated that modafinil modulates astroglial connexin-based gap junctional communication as administration of a low dose of flecainide, an astroglial connexin inhibitor, enhanced the wake-promoting and procognitive activity of modafinil in rodents and healthy volunteers. The aim of this study is to investigate changes in glucose cerebral metabolism in rodents, induced by the combination of modafinil+ flecainide low dose (called THN102). Methods: The impact of THN102 on brain glucose metabolism was noninvasively investigated using F-2-fluoro-2-deoxy-D- glucose Positron Emission Tomography imaging in Sprague-Dawley male rats. Animals were injected with vehicle, flecainide, modafinil, or THN102 and further injected with F-2-fluoro-2-deoxy-D-glucose followed by 60-minute Positron Emission Tomography acquisition. F-2-fluoro-2-deoxy-D-glucose Positron Emission Tomography images were coregistered to a rat brain template and normalized from the total brain Positron Emission Tomography signal. Voxel-to-voxel analysis was performed using SPM8 software. Comparison of brain glucose metabolism between groups was then performed. Results: THN102 significantly increased regional brain glucose metabolism as it resulted in large clusters of F-2-fluoro- 2-deoxy-D-glucose uptake localized in the cortex, striatum, and amygdala compared with control or drugs administered alone. These regions, highly involved in the regulation of sleep-wake cycle, emotions, and cognitive functions were hence quantitatively modulated by THN102. Conclusion: Data presented here provide the first evidence of a regional brain activation induced by THN102, currently being tested in a phase II clinical trial in narcoleptic patients. Keywords: modafinil, astroglial connexin, FDG PET imaging, neuroglia, narcolepsy Received: November 17, 2017; Revised: February 21, 2018; Accepted: March 14, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any 687 medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 688 | International Journal of Neuropsychopharmacology, 2018 Significance Statement THN102 (modafinil/flecainide) is efficient to reduce excessive daytime sleepiness. The impact of THN102 on rat brain metabo- lism was investigated with FDG PET imaging. Cortex, striatum, and amygdala were more activated after THN102 compared with modafinil. This study provides insights on the mechanism of action of THN102 innovative product. Introduction Modafinil is a nonamphetaminic, wake-promoting compound The study was conducted in accordance with the French legisla- used as first line treatment of excessive daytime sleepiness tion and European directives on the use of animals in research (EDS), associated with narcolepsy (Lavault et  al., 2011 Thorp ; y (EU Directive 2010/63/EU). The protocol has been approved by and Dauvilliers, 2015; Barateau et  al., 2016). It has also been the local committee for animal use in research (APAFIS#7466-20 proposed as a treatment to reduce EDS in Parkinson’s disease 1611 04 1 7049220 v2). (Sheng et al., 2013; Rodrigues et al., 2016). Mechanisms involved in wake-promoting and procognitive actions of modafinil Drugs and Chemicals are complex, as it modulates multiple monoaminergic and Drugs were administered by i.v. injection in the tail vein (1 mL/kg). GABAergic neuronal systems (Minzenberg and Carter, 2008). Treatments included vehicle (VEH), modafinil 10  mg/kg (MOD; Early studies using Fos as a marker of neuronal activity in Orchid Pharma), flecainide 1 mg/kg (FLE; Sigma-Aldrich), or the cat (Lin et  al., 1996) and rat (Engber et  al., 1998) suggested the combination of both (THN102). FDG for i.v. injection was pur - hypothalamus as main brain target of modafinil. Subsequent chased from Cyclopharma. studies in rodents indicated that cortex and striatum were activated after wake enhancement by modafinil (Scammell et  al., 2000; Willie et  al., 2005). Imaging in rodents depicted FDG PET Imaging: Acquisition Protocol and Imaging higher metabolism in hippocampus, thalamus, and amygdala Data Analysis (Engber et al., 1998) and showed activating effects of modafinil PET imaging study was performed using PET systems coupled in fronto-cortical areas (Gozzi et  al., 2012); those areas are with a computerized tomography scanner (Inveon microPET-CT; involved in both arousal (Duteil et al., 1990 Lin et  ; al., 1992) and spatial resolution ~1.6 mm; Siemens) in anesthetized and fasted cognitive enhancement (Lynch et  al.; Béracochéa et  al., 2003). rats (1.5%–2.5% inhaled isoflurane, weight 250–350 g) (Bao et al., Using pharmacological magnetic resonance imaging, or sur - 2009). PET experiments were exclusively performed in the morn- face electroencephalography, modafinil was found to increase ing. Each day of experiment, 4 rats were randomly assigned to brain activity notably in the hippocampus and frontal cortex in each group: animals were i.v. injected with VEH, MOD, FLE, or healthy volunteers (Joo et  al., 2008a) and narcoleptic patients THN102 before being placed into the scanner for computer - (Saletu et al., 2007J; oo et al., 2008b). Moreover, positron emission ized tomography acquisition. Thirty minutes after the admin- tomography (PET) using F-2-fluoro-2-deoxy-D-glucose (FDG) in istration, 1  mL FDG (mean dose = 42.2 ± 26.3 MBq) was injected narcoleptic patients unveiled that modafinil increased the glu- over 1 minute using a syringe pump. Dynamic PET acquisition cose brain metabolism in the hippocampus (Kim et al., 2007). begun immediately after the start of FDG infusion for 60 min- More recently, modafinil was shown to modulate astrocyte utes. Blood glucose measurement was performed before F-FDG functions by increasing cell-cell communication mediated injection and at the end of PET acquisition using a portable glu- by connexin channels (Liu et  al., 2013), membrane proteins cometer (Accu-check Performa, Roche). involved in cellular communication (Giaume et  al., 2010), and PET data were reconstructed using the FORE + OSEM2D sleep regulation (Franco-Pérez and Paz, 2009F ; ranco-Pérez et al., algorithm including normalization, attenuation, scatter, and 2012; Clasadonte et  al., 2017). It was further shown in rodents random corrections. To reduce noise and correct for partial vol- that modulating astrocyte connexin by flecainide impacted the ume effect, an iterative deconvolution using the point spread pharmacological action of modafinil by enhancing its wake- function of the scanner with a temporal based denoising was promoting, procognitive, and, notably, antinarcoleptic effects applied to each image. The method was previously reported and (Duchêne et  al., 2016; Lu and Chen, 2016). The mechanisms of validated for use in small animal PET imaging (Wimberley et al., action of the modafinil/flecainide combination (THN102) remain 2014; Reilhac et al., 2015). to be further investigated but would likely be based on a res- Dynamic and summed (30–60 minutes) FDG PET images were toration of the functionality of astroglial connexins. Using FDG spatially normalized to a standard rat brain FDG Schiffer’s tem- PET in the rat, the aim of the present study was to assess the plate using Pmod software (version 3.6) (Schiffer et al., 2006). The CNS effects of THN102 at the functional level compared with brain kinetics of FDG may depend on its plasma kinetics (input modafinil or flecainide used alone, both at their effective and function) and peripheral blood glucose level. Two normalization clinically relevant doses. methods were thus performed to detect any regional change in FDG uptake by the brain. First, summed PET images were nor- Materials and Methods malized by their respective whole-brain activity, thus highlight- ing the relative FDG uptake by the different brain regions. Then, Animals the absolute metabolic rate of glucose (MRGlu) was estimated Male Sprague-Dawley rats (Elevage Janvier) were collectively using pharmacokinetic modelling. To that end, a volume of housed with food and water ad libitum. They were kept in a interest was drawn on the vena cava to generate an imaged- temperature- and humidity-controlled facility with 12-hour- derived input function of FDG, as previously described (Weber dark/-light cycles (lights on at 8:00 am). Experiments were per - et  al., 2002; Lanz et  al., 2014). MRGlu Parametric PET images formed during the light phase between 9:00 am and 1:00 pm. (PXMOD, Pmod software, version 3.6) were then generated for Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 Vodovar et al. | 689 each animal from the dynamic brain PET images and corre- THN102 Locally Increases the Brain Glucose sponding imaged-derived input function (from 0 to 60 minutes) Metabolism using the FDG Patlak model, considering blood glucose level Normalization of FDG uptake by whole-brain activity was associ- measured immediately before PET (Patlak et al., 1983). The 0.71 ated with a low variability. In the VEH group, the coefficient of lumped constant was used to take the difference between glu- variation (CV = SD/mean × 100) of the relative uptake of FDG was cose and FDG metabolism into acount (Tokugawa et al., 2007). CV = 2.73% in the cortex. SPM analysis did not show any signifi- Comparison of parametric images (FDG brain uptake and cant difference in the distribution of relative FDG brain uptake MRGlu) obtained in each group (n= 5 animals/group) was per- between FLE or MOD groups compared with the VEH group formed using a statistical parametric mapping (SPM) and (P > .05). Significant increase in the relative brain uptake of FDG a voxel-to-voxel analysis (SPM8 softwared) as previously could be regionally observed in the THN102 group compared described (Schiffer et  al., 2006; Soto-Montenegro et  al., 2009). with the MOD group. The effect was observed bilaterally and was A  brain mask was created from the FDG template and applied homogenously distributed in the cortex, amygdala and striatum to all registered and normalized scans to include only cere- (Table 1; Figure 1). Notably, a cluster of significant increase in FDG bral voxels. Comparison was then performed using an ANOVA uptake could be observed in the nucleus accumbens. Similar dis- design to detect differences between groups. A significance level tribution of increased relative FDG uptake could be observed in threshold of .05 (uncorrected for multiple comparisons) and a the THN102 group compared with the FLE or MOD alone (P < .05). minimum cluster size of 200 voxels were selected. Only the clus- Within the significant clusters, further detailed analysis ters that were significant at P < .05 levels (corrected for multiple allowed to locate the relevant peak regions. Hence, THN102 comparisons) were considered. The size of the clusters exceed- enhanced the brain metabolism compared with MOD in the ing the threshold and their corrected significance were ana- subsortical regions such as the dysgranular and retrospleni- tomically located using the Paxinos and Watson rat brain atlas aldysgranular cortices, primary/secondary motor, dorsolateral (Paxinos and Watson, 2007). enthorinal and visual cortices, the retrosplenialdysgranular area, as well as the layer 3 and basolateral amygdaloïd nuclei. Spontaneous Locomotor Activity Assessment There was a significant increase in blood glucose levels meas- ured before (85.8 ± 4.5 mg/dL) and at the end (116± 4.3 mg/dL) of In an independent experiment, spontaneous locomotor activ- PET acquisitions (P < .05). However, initial as well as final glucose ity was recorded in an open-field device (50 x 50 x 50 cm), during concentrations were not different between the 4 treament groups, 2 hours, starting immediately after administration (VEH, MOD, suggesting the absence of treatement-induced change in periph- FLE, and THN102). Cumulated traveled distance per 5-minute eral glucose metabolism. Compared with the relative FDG uptake, time bins was analyzed with ViewPoint. Experiments were per - absolute quantification of the brain MRGlu was associated with formed with 8 drug-naïve animals in each group. The experi- a higher variability (CV = 19.82% in the cortex of the VEH group). menter was blinded to treatment. SPM analysis nonetheless highlighted patterns of significantly higher glucose consumption in the THN102 group compared Determination of Modafinil Concentration in Serum with MOD alone (P < .05). The regional increase in MRGlu was and Brain consistent with the regional increase in the relative FDG uptake (Table  2; Figure  2). However, difference in MRGlu in the THN102 In parallel experiments, performed in another population of group compared with the VEH and FLE groups was not significant, rats, brain and serum concentrations of modafinil were deter - which may be due to a higher variability of the MRGlu data. mined after MOD and THN102 administration in rats. Animals (n = 8 rats/group) were anesthetized with isoflurane 30 minutes after i.v. administration and blood samples were collected, cen- THN102 Increases Spontaneous Locomotor Activity trifuged (3000 g× 15 min, 20°C), and stored at -80°C. Brains were To assess whether doses of modafinil allowed to discriminate collected immediately after blood sampling, freezed on dry ice, THN102 compared with modafinil alone, spontaneous motor and stored at -80°C. Modafinil and flecainide concentrations in activity in unrestrained awake rats was monitored for 2 hours after serum and brain lysate were determined using a reverse phase treatment, during the lights-on period (Figure 3). Locomotor activ- liquid chromatography with tandem mass spectrometry detec- ity in the MOD group tends to be increased compared with the tion technique (Eurofins ADME Bioanalyses). VEH group without reaching significance (+15.2% at 120 minutes). THN102 administration induced a significant increase in cumu- Statistical Analysis lative locomotor activity compared with VEH-treated animals, beginning at 95 minutes (+22.0% at 120 minutes vs VEH,P = .0309). Results of the locomotion and pharmacokinetic data are THN102 was more effective in increasing the locomotor activity expressed as mean ± SEM. Difference was considered signifi- compared with MOD; however, the difference was not statistically cant at P< .05 levels. Statistical analysis was performed using significant (+5.89% at 120 minutes). Moreover, no effect of FLE treat- Graphpad software (GraphPad Prism version 7). Cumulative ment was found at the 1-mg/kg dose when administered alone. traveled distance over time was compared using a 2-way repeated-measure ANOVA followed by Bonferroni’s posthoc test. Finally, serum and brain modafinil concentrations were com- Brain and Serum Concentrations of Modafinil pared using an unpaired t test. Serum and brain were collected 30 minutes after treatment with modafinil alone (MOD) or combined with flecainide (THN102; Results Figure  4). Quantification of modafinil concentration levels The goal of this study was to compare the brain metabolism treated with MOD or THN102 demonstrated no significant dif- after treatment with THN102, combination between modafinil ference between both groups in serum (2.12± 0.472  ng/mL and and flecainide at low dose, to modafinil alone. 2.14 ± 0.419 ng/mL, respectively) or in brain (1.37± 0.262 ng/g and Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 690 | International Journal of Neuropsychopharmacology, 2018 Table  1. SPM Results Showing Clusters, Peak Coordinates, and Significant Levels of Brain Regions with an Increase in F-FDG Uptake When Comparing THN102 to MOD, FLE, or VEH Significant Peak Coordinates within Clusters Intergroup Comparisons Significant Clusters x y z Peak Regions THN102 > VEH Cortex 0.5 4.6 3.6 R pre-limbic cortex Caudate and putamen (Striatum) 6.7 4.6 -5.6 R primary auditory cortex Amygdala -5.7 3 -8.8 L temporal association cortex -0.1 1.2 -0.2 L cingulate cortex area 1 -5.5 8.2 -0.6 L layer 3 of cortex THN102 > FLE Cortex 6.5 4.6 -5.8 R primary auditory cortex Caudate and putamen (Striatum) 4.3 2.8 2.0 R primary somatosensory Amygdala -5.5 8.6 -4.8 L layer 3 of cortex -2.1 3.6 3.8 L forceps minor corpus callosum -3.1 0.4 -7.8 L primary visual cortex monocular THN102 > MOD Cortex 5.7 7.2 -1.6 R dysgranular cortex Caudate and putamen (Striatum) 2.1 1.4 4.4 R secondary motor cortex Amygdala 2.5 0.8 -5.2 R secondary visual cortex -0.7 0.4 -4.2 L retrosplenialdysgranular cortex -3.7 6.8 3.0 L layer 3 of cortex -5.5 8.6 -4.8 L basolateral amygdaloïd nucleus, posterior Voxels comparison-based analysis was performed using SPM8 (n = 5) rats per group using 1-way ANOVA followed by multiple comparison test. A significance level threshold of .05 and a minimum cluster size of 200 voxels were selected to identify significant clusters. Coordinates of sig- nificant peak (P < .05) were given according to the Paxinos and Watson rat brain atlas (Paxinos and Watson, 2007). FLE, flecainide 1 mg/kg; MOD, modafinil 10 mg/kg; THN102, modafinil 10 mg/kg + flecainide 1 mg/kg; VEH, vehicle. 18 18 Figure 1. Coronal brain section showing statistical parametric map for increase in relativ F-2-fluor e o-2-deoxy-D-glucose ( F-FDG) uptake after Modafinil (MOD) + fle- cainide (THN102) treatment in comparison to MOD alone in rats. Following a cranio-caudal orientation centered on bregma (anterior is positive), coronal brain sections were at (a) 1.05, (b) -0.68, (c) -1.29, (d) -3.07, (e) -4.34, and (f) -5.76 millimeters from the bregma and (g) sagittal view of the brain with the marked coronal sections (red dotted lines) from a to f. Color scale represents all T distributions achieving statistical significance (see SPM statistical analysis). Acb, accumbens; Amy, amygdala; Cg, cingulate cortex; Ent, entorhinal cortex; Ic, insular cortex; M, motor cortex; RS, retrosplenial cortex; SS, somatosensory cortex; Str, striatum; V, visual cortex. 1.50 ± 0.277  ng/g, respectively). Therefore, there is no apparent Modafinil has been largely proposed in sleep medicine to pharmacokinetic interaction between MOD and FLE on MOD treat EDS associated with narcolepsy (Lavault et al., 2011 Thorp ; y metabolism. and Dauvilliers, 2015; Barateau et al., 2016), Parkinson’s disease (Sheng et  al., 2013; Rodrigues et  al., 2016), idiopathic hyper - somnia (Lavault et  al., 2011; Lopez et  al., 2017), and obstruct- Discussion ive sleep apnea/hypopnea syndrome (Black and Hirshkowitz, In this study, we reported that THN102 significantly activates 2005). Numerous preclinical studies have generated a wealth of experimental data, which lead to many hypotheses regard- the cortico-amygdala-striata regions compared with MOD used alone. ing the mode of action of modafinil (Gerrard and Malcolm, 2007; Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 Vodovar et al. | 691 Table 2. SPM Results Showing Clusters, Peak Coordinates, and Significant Levels of Brain Regions with an Increase in MRGlu When Comparing THN102 to MOD in Rats Significant Peak Coordinates within Clusters Intergroup Comparisons Significant Clusters x y z Peak Regions THN102 > MOD Cortex 4.1 0.8 -5.2 R primary visual cortex Caudate and putamen (Striatum) 0.7 2.2 3.8 R secondary motor cortex Amygdala 5.7 8.6 -4.6 R layer 3 of cortex Accumbens -1.5 0.6 -0.4 L primary motor cortex -4.9 9.0 0.0 L layer 3 of cortex -6.5 6.4 -8.8 L dorsolateral enthorinal cortex Voxels comparison based analysis was performed using SPM8 (n = 5) rats per group using 1-way ANOVA followed by multiple comparison test. A  significance level threshold of .05 and a minimum cluster size of 200 voxels were selected to identify significant clusters. Coordinates of significant peak (P < .05) were given according to the Paxinos and Watson rat brain atlas (Paxinos and Watson, 2007). MOD, modafinil 10 mg/kg; MRGlu, metabolic rate of glucose; THN102, modafinil 10 mg/kg + flecainide 1 mg/kg. Figure  2. Coronal brain section showing statistical parametric map for increase in absolute metabolic rate of glucose (MRGlu) after Modafinil (MOD) + flecainide (THN102) treatment in comparison to MOD in rats. Following a cranio-caudal orientation centered on bregma (anterior is positive), coronal brain sections were at (a) 1.05, (b) -0.68, (c) -1.29, (d) -3.07, (e) -4.34, and (f) -5.76 millimeters from the bregma; and (g) sagittal view of the brain with the marked coronal sections (red dotted lines) from a to f. Color scale represents all T distributions achieving statistical significance (see SPM statistical analysis). Acb, accumbens; Amy, amygdala; Cg, cingulate cor - tex; Ent, entorhinal cortex; Ic, insular cortex; M, motor cortex; RS, retrosplenial cortex; SS, somatosensory cortex; Str, striatum; V, visual cortex. Minzenberg and Carter, 2008). The central noradrenergic Recent data indicated that not only neurons but also glial cells, in hypothesis has been supported by data showing that inhibition particular astrocytes, were modulated by modafinil, as it enhanced of catecholamine synthesis or antagonism of adrenergic recep- astrocyte coupling and the expression of one of their connexins, tors is able to attenuate the wake-promoting effects of modafinil Cx30 (Liu et al., 2013). These data suggest that astroglial connexins (Duteil et  al., 1991; Lin et  al., 1992). Recent studies using mice might be involved in modafinil mode of action. Indeed, flecainide, lacking the noradrenaline synthesis or alpha1β-adrenoceptor an astroglial connexin inhibitor, was able to enhance modafinil (Stone et al., 2002a, 2002b; Hou et al., 2005) or brain imaging in procognitive and wake-promoting activities when coadministered humans (Minzenberg and Carter, 2008) also support the crit- to modafinil in rodents (Duchêne et al., 2016; Lu and Chen, 2016). ical involvement of the locus cœruleus noradrenergic system in THN102 is currently in phase II clinical trial on narcoleptic patients modafinil profile. The dopaminergic hypothesis has been very (NCT02821715). In this context, we compared the impact on brain attractive and prevailing since the identification of an affinity of functions between THN102 and modafinil in the rat by assessing modafinil with dopamine transporter (Mignot et al., 1994; Wisor their drug-induced changes in brain glucose metabolism. et al., 2001; Wisor and Eriksson, 2005; Korotkova et al., 2007Qu ; The route of administration and dose of modafinil et al., 2008). The brain disinhibitory hypothesis (Lin et al., 1996, (10 mg/kg, i.v.) was based on a previous functional magnetic res- 2000) has received less attention, yet it is supported by the fact onance imaging (fMRI) study in rats (Gozzi et al., 2012). Flecainide that modafinil induces a significant decrease in GABA outflow dose was chosen according to previous studies in rodents (Ferraro et al., 1996) in many brains areas, notably those critic- (Duchêne et al., 2016). Additionally, at selected doses, THN102 sig- ally involved in sleep-wake cycle control. nificantly increased locomotor activity, whereas modafinil alone Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 692 | International Journal of Neuropsychopharmacology, 2018 Figure 3. Cumulative traveled distance over 2 hours after vehicle (VEH), flecainide 1 mg/kg (FLE), modafinil 10 mg/kg (MOD), or MOD 10 mg/kg + FLE 1 mg/kg (THN102) treatment in awake rats. Locomotor activity of rats was measured during 2 hours after treatment. Data are expressed as means ± SEM (n = 8 rats/group) and compared using 2-way ANOVA followed by Bonferroni’s multiple comparison test: *P < .05 THN102 vs VEH. Figure 4. Serum (a) and brain (b) concentrations of modafinil after modafinil 10 mg/kg + flecainide 1 mg/kg (THN102) and modafinil 10 mg/kg (MOD) treatment. Thirty minutes after the treatment injection, serum and brain levels of MOD were sampled and quantified by liquid chromatography with tandem mass spectrometry. Data are expressed as means with scattered plots ± SEM (n = 8 rats/group) and compared using an unpaired t test. showed only a tendency to increase it (nonsignificant), as partially Gozzi et  al., 2012), including fMRI, 2-deoxyglucose autoradi- described elsewhere (Simon et  al., 1996; Edgar and Seidel, 1997; ography, FDG PET, and cerebral blood flow assesement using Zolkowska et al., 2009). As previously reported in mice (Duchêne single-photon emission computed tomography. These data et al., 2016), assessment of modafinil concentration in the serum tend towards a consensus over cortical and subcortical brain and brain confirmed that flecainide did not increase the brain activations induced by modafinil including hypothalamic and distribution of modafinil. Therefore, pharmacokinetic interaction thalamic regions (Thomas and Kwong, 2006; Gozzi et al., 2012), between flecainide and modafinil can unlikely explain the brain which are highly involved in the regulation of sleep-wake cycle effect of THN102 compared with vehicle and modafinil alone. (Szabadi, 2006; Lin et al., 2011). Other major structures are high- Serum concentrations were close to modafinil levels found in lighted following modafinil administration as the caudate puta- subjects following dosing of clinically effective doses (McClellan men (striatum), the amygdala, and the hippocampus (Engber and Spencer, 1998; Wong et  al., 1999), suggesting potential rele- et al., 1998; Ghahremani et al., 2011), known for their implication vance of the presented data to clinical conditions. in driving emotions and cognitive functions (Joo et al., 2008a). Several methods have been proposed to investigate the CNS In our conditions, modafinil alone did not modulate glucose effects of modafinil in human (Ellis et  al., 1999; Spence et  al., brain metabolism assessed by FDG PET compared with vehicle. 2005; Hunter et  al., 2006; Thomas and Kwong, 2006; Kim et  al., Even though the dosage and the route were identical to a previ- 2007; Joo et al., 2008a; Minzenberg et al., 2008; Rasetti et al., 2010; ous fMRI study (Gozzi et  al., 2012), the observed cortical acti- Ghahremani et  al., 2011; Minzenberg et  al., 2011; Goudriaan vation after treatment was not detected in our analysis. This et al., 2013; Schmaal et al., 2013; Funayama et al., 2014; Schmaal difference highlights the discrepancies that may exist between et al., 2014; Ikeda et al., 2017; Schmidt et al., 2017) and animals the drug-induced response on blood oxygenation levels- (Engber et  al., 1998; van Vliet et  al., 2008; Dawson et  al., 2012; dependent response and on energy consumption (Di et al., 2012; Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 Vodovar et al. | 693 Cabrera et  al., 2016). Another explanation for such discrepan- sleep-wake cycle and behaviors. Our results further support cies may be the presence of anesthesia. In the present study, the hypothesis that astrocyte connexins are involved in phar - animals were exposed to <2.5% isoflurane during drug admin- macological responses of psychoactive drugs such as modafinil istration and subsequent PET acquisition. It was reported that (Duchêne et al., 2016; Jeanson et al., 2016; Charvériat et al., 2017). isoflurane may modulate the brain glucose uptake and may thus limit the sensitivity of the method to detect the CNS response Funding to investigated compounds in vivo (Spangler-Bickell et al., 2016; Park et al., 2017). Working on awake rodents was shown feasible, This work was supported by Theranexus Company and the although technically challenging (Spangler-Bickell et  al., 2016; Agence Nationale de la Recherche (grant number 14-CE16-0022). Park et  al., 2017). In the present study, we chose to administer investigated treatments under isoflurane anesthesia to facilitate Acknowledgments animal handling, i.v. administration, and avoid any stress due to the experimental procedure. Moreover, we showed that the loco- We thank Emile Jaumain for helpful technical assistance. motor activity was different between groups, which may non- specifically impact FDG uptake by the brain in awake animals. Statement of Interest Therefore, the whole procedure was performed under isoflurane anesthesia to highlight the intrinsic effect of each treatment on F.M., M.C., and A.D. are full-time employees of Theranexus com- brain function and allow for dynamic PET acquisition for 60 pany. Y.D.  served as consultant for Bioprojet Pharma, Flamel minutes in immobile animals. Technologies, Jazz Pharmaceuticals, Theranexus, Takeda, and Some brain structures such as the locus coeruleus, thala- UCB. The other authors declare no financial conflict of interest. mus, and hypothalamus, modulated by modafinil (Minzenberg et  al., 2008; Gozzi et  al., 2012; Schmaal et  al., 2013) were not References activated by THN102 compared with modafinil alone. More Bao Q, Newport D, Chen M, Stout DB, Chatziioannou AF (2009) interestingly, this study reported that THN102 significantly Performance evaluation of the inveon dedicated PET preclin- increased the relative glucose brain uptake in the whole cortex, ical tomograph based on the NEMA NU-4 standards. J Nucl amygdala, and striatum compared with modafinil alone, hence Med 50:401–408. suggesting a different activity on glucose metabolism between Barateau L, Lopez R, Dauvilliers Y (2016) Treatment options for both treatment groups. Similar effects could be observed using narcolepsy. CNS Drugs 30:369–379. pharmacokinetic modelling and estimation of the absolute Bayard S, Croisier Langenier M, Cochen De Cock V, Scholz S, MRGlu. Despite higher variability in this parameter, patterns of Dauvilliers Y (2012) Executive control of attention in narco- increased metabolic rate of glucose were detected in the cortex, lepsy. Plos One 7:e33525. striatum, amygdala, and accumbens of rats in the THN102 group Béracochéa D, Celerier A, Peres M, Pierard C (2003) Enhancement compared with the MOD group. Such brain regions may be con- of learning processes following an acute modafinil injection sidered as substrates for the enhancement of modafinil effects in mice. Pharmacol Biochem Behav 76:473–479. by flecainide. They notably process attention states and motiva- Black JE, Hirshkowitz M (2005) Modafinil for treatment of tion and code behavioral responses (Cardinal et  al., 2002 Cho ; residual excessive sleepiness in nasal continuous positive et al., 2013), functions that are altered in narcolepsy and that are airway pressure-treated obstructive sleep apnea/hypopnea portentially modulated by THN102 (Bayard et al., 2012). syndrome. Sleep 28:464–471. 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Cortico-Amygdala-Striatal Activation by Modafinil/Flecainide Combination

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Abstract

Background: Modafinil, a nonamphetaminic wake-promoting compound, is prescribed as first line therapy in narcolepsy, an invalidating disorder characterized by excessive daytime sleepiness and cataplexy. Although its mode of action remains incompletely known, recent studies indicated that modafinil modulates astroglial connexin-based gap junctional communication as administration of a low dose of flecainide, an astroglial connexin inhibitor, enhanced the wake-promoting and procognitive activity of modafinil in rodents and healthy volunteers. The aim of this study is to investigate changes in glucose cerebral metabolism in rodents, induced by the combination of modafinil+ flecainide low dose (called THN102). Methods: The impact of THN102 on brain glucose metabolism was noninvasively investigated using F-2-fluoro-2-deoxy-D- glucose Positron Emission Tomography imaging in Sprague-Dawley male rats. Animals were injected with vehicle, flecainide, modafinil, or THN102 and further injected with F-2-fluoro-2-deoxy-D-glucose followed by 60-minute Positron Emission Tomography acquisition. F-2-fluoro-2-deoxy-D-glucose Positron Emission Tomography images were coregistered to a rat brain template and normalized from the total brain Positron Emission Tomography signal. Voxel-to-voxel analysis was performed using SPM8 software. Comparison of brain glucose metabolism between groups was then performed. Results: THN102 significantly increased regional brain glucose metabolism as it resulted in large clusters of F-2-fluoro- 2-deoxy-D-glucose uptake localized in the cortex, striatum, and amygdala compared with control or drugs administered alone. These regions, highly involved in the regulation of sleep-wake cycle, emotions, and cognitive functions were hence quantitatively modulated by THN102. Conclusion: Data presented here provide the first evidence of a regional brain activation induced by THN102, currently being tested in a phase II clinical trial in narcoleptic patients. Keywords: modafinil, astroglial connexin, FDG PET imaging, neuroglia, narcolepsy Received: November 17, 2017; Revised: February 21, 2018; Accepted: March 14, 2018 © The Author(s) 2018. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any 687 medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 688 | International Journal of Neuropsychopharmacology, 2018 Significance Statement THN102 (modafinil/flecainide) is efficient to reduce excessive daytime sleepiness. The impact of THN102 on rat brain metabo- lism was investigated with FDG PET imaging. Cortex, striatum, and amygdala were more activated after THN102 compared with modafinil. This study provides insights on the mechanism of action of THN102 innovative product. Introduction Modafinil is a nonamphetaminic, wake-promoting compound The study was conducted in accordance with the French legisla- used as first line treatment of excessive daytime sleepiness tion and European directives on the use of animals in research (EDS), associated with narcolepsy (Lavault et  al., 2011 Thorp ; y (EU Directive 2010/63/EU). The protocol has been approved by and Dauvilliers, 2015; Barateau et  al., 2016). It has also been the local committee for animal use in research (APAFIS#7466-20 proposed as a treatment to reduce EDS in Parkinson’s disease 1611 04 1 7049220 v2). (Sheng et al., 2013; Rodrigues et al., 2016). Mechanisms involved in wake-promoting and procognitive actions of modafinil Drugs and Chemicals are complex, as it modulates multiple monoaminergic and Drugs were administered by i.v. injection in the tail vein (1 mL/kg). GABAergic neuronal systems (Minzenberg and Carter, 2008). Treatments included vehicle (VEH), modafinil 10  mg/kg (MOD; Early studies using Fos as a marker of neuronal activity in Orchid Pharma), flecainide 1 mg/kg (FLE; Sigma-Aldrich), or the cat (Lin et  al., 1996) and rat (Engber et  al., 1998) suggested the combination of both (THN102). FDG for i.v. injection was pur - hypothalamus as main brain target of modafinil. Subsequent chased from Cyclopharma. studies in rodents indicated that cortex and striatum were activated after wake enhancement by modafinil (Scammell et  al., 2000; Willie et  al., 2005). Imaging in rodents depicted FDG PET Imaging: Acquisition Protocol and Imaging higher metabolism in hippocampus, thalamus, and amygdala Data Analysis (Engber et al., 1998) and showed activating effects of modafinil PET imaging study was performed using PET systems coupled in fronto-cortical areas (Gozzi et  al., 2012); those areas are with a computerized tomography scanner (Inveon microPET-CT; involved in both arousal (Duteil et al., 1990 Lin et  ; al., 1992) and spatial resolution ~1.6 mm; Siemens) in anesthetized and fasted cognitive enhancement (Lynch et  al.; Béracochéa et  al., 2003). rats (1.5%–2.5% inhaled isoflurane, weight 250–350 g) (Bao et al., Using pharmacological magnetic resonance imaging, or sur - 2009). PET experiments were exclusively performed in the morn- face electroencephalography, modafinil was found to increase ing. Each day of experiment, 4 rats were randomly assigned to brain activity notably in the hippocampus and frontal cortex in each group: animals were i.v. injected with VEH, MOD, FLE, or healthy volunteers (Joo et  al., 2008a) and narcoleptic patients THN102 before being placed into the scanner for computer - (Saletu et al., 2007J; oo et al., 2008b). Moreover, positron emission ized tomography acquisition. Thirty minutes after the admin- tomography (PET) using F-2-fluoro-2-deoxy-D-glucose (FDG) in istration, 1  mL FDG (mean dose = 42.2 ± 26.3 MBq) was injected narcoleptic patients unveiled that modafinil increased the glu- over 1 minute using a syringe pump. Dynamic PET acquisition cose brain metabolism in the hippocampus (Kim et al., 2007). begun immediately after the start of FDG infusion for 60 min- More recently, modafinil was shown to modulate astrocyte utes. Blood glucose measurement was performed before F-FDG functions by increasing cell-cell communication mediated injection and at the end of PET acquisition using a portable glu- by connexin channels (Liu et  al., 2013), membrane proteins cometer (Accu-check Performa, Roche). involved in cellular communication (Giaume et  al., 2010), and PET data were reconstructed using the FORE + OSEM2D sleep regulation (Franco-Pérez and Paz, 2009F ; ranco-Pérez et al., algorithm including normalization, attenuation, scatter, and 2012; Clasadonte et  al., 2017). It was further shown in rodents random corrections. To reduce noise and correct for partial vol- that modulating astrocyte connexin by flecainide impacted the ume effect, an iterative deconvolution using the point spread pharmacological action of modafinil by enhancing its wake- function of the scanner with a temporal based denoising was promoting, procognitive, and, notably, antinarcoleptic effects applied to each image. The method was previously reported and (Duchêne et  al., 2016; Lu and Chen, 2016). The mechanisms of validated for use in small animal PET imaging (Wimberley et al., action of the modafinil/flecainide combination (THN102) remain 2014; Reilhac et al., 2015). to be further investigated but would likely be based on a res- Dynamic and summed (30–60 minutes) FDG PET images were toration of the functionality of astroglial connexins. Using FDG spatially normalized to a standard rat brain FDG Schiffer’s tem- PET in the rat, the aim of the present study was to assess the plate using Pmod software (version 3.6) (Schiffer et al., 2006). The CNS effects of THN102 at the functional level compared with brain kinetics of FDG may depend on its plasma kinetics (input modafinil or flecainide used alone, both at their effective and function) and peripheral blood glucose level. Two normalization clinically relevant doses. methods were thus performed to detect any regional change in FDG uptake by the brain. First, summed PET images were nor- Materials and Methods malized by their respective whole-brain activity, thus highlight- ing the relative FDG uptake by the different brain regions. Then, Animals the absolute metabolic rate of glucose (MRGlu) was estimated Male Sprague-Dawley rats (Elevage Janvier) were collectively using pharmacokinetic modelling. To that end, a volume of housed with food and water ad libitum. They were kept in a interest was drawn on the vena cava to generate an imaged- temperature- and humidity-controlled facility with 12-hour- derived input function of FDG, as previously described (Weber dark/-light cycles (lights on at 8:00 am). Experiments were per - et  al., 2002; Lanz et  al., 2014). MRGlu Parametric PET images formed during the light phase between 9:00 am and 1:00 pm. (PXMOD, Pmod software, version 3.6) were then generated for Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 Vodovar et al. | 689 each animal from the dynamic brain PET images and corre- THN102 Locally Increases the Brain Glucose sponding imaged-derived input function (from 0 to 60 minutes) Metabolism using the FDG Patlak model, considering blood glucose level Normalization of FDG uptake by whole-brain activity was associ- measured immediately before PET (Patlak et al., 1983). The 0.71 ated with a low variability. In the VEH group, the coefficient of lumped constant was used to take the difference between glu- variation (CV = SD/mean × 100) of the relative uptake of FDG was cose and FDG metabolism into acount (Tokugawa et al., 2007). CV = 2.73% in the cortex. SPM analysis did not show any signifi- Comparison of parametric images (FDG brain uptake and cant difference in the distribution of relative FDG brain uptake MRGlu) obtained in each group (n= 5 animals/group) was per- between FLE or MOD groups compared with the VEH group formed using a statistical parametric mapping (SPM) and (P > .05). Significant increase in the relative brain uptake of FDG a voxel-to-voxel analysis (SPM8 softwared) as previously could be regionally observed in the THN102 group compared described (Schiffer et  al., 2006; Soto-Montenegro et  al., 2009). with the MOD group. The effect was observed bilaterally and was A  brain mask was created from the FDG template and applied homogenously distributed in the cortex, amygdala and striatum to all registered and normalized scans to include only cere- (Table 1; Figure 1). Notably, a cluster of significant increase in FDG bral voxels. Comparison was then performed using an ANOVA uptake could be observed in the nucleus accumbens. Similar dis- design to detect differences between groups. A significance level tribution of increased relative FDG uptake could be observed in threshold of .05 (uncorrected for multiple comparisons) and a the THN102 group compared with the FLE or MOD alone (P < .05). minimum cluster size of 200 voxels were selected. Only the clus- Within the significant clusters, further detailed analysis ters that were significant at P < .05 levels (corrected for multiple allowed to locate the relevant peak regions. Hence, THN102 comparisons) were considered. The size of the clusters exceed- enhanced the brain metabolism compared with MOD in the ing the threshold and their corrected significance were ana- subsortical regions such as the dysgranular and retrospleni- tomically located using the Paxinos and Watson rat brain atlas aldysgranular cortices, primary/secondary motor, dorsolateral (Paxinos and Watson, 2007). enthorinal and visual cortices, the retrosplenialdysgranular area, as well as the layer 3 and basolateral amygdaloïd nuclei. Spontaneous Locomotor Activity Assessment There was a significant increase in blood glucose levels meas- ured before (85.8 ± 4.5 mg/dL) and at the end (116± 4.3 mg/dL) of In an independent experiment, spontaneous locomotor activ- PET acquisitions (P < .05). However, initial as well as final glucose ity was recorded in an open-field device (50 x 50 x 50 cm), during concentrations were not different between the 4 treament groups, 2 hours, starting immediately after administration (VEH, MOD, suggesting the absence of treatement-induced change in periph- FLE, and THN102). Cumulated traveled distance per 5-minute eral glucose metabolism. Compared with the relative FDG uptake, time bins was analyzed with ViewPoint. Experiments were per - absolute quantification of the brain MRGlu was associated with formed with 8 drug-naïve animals in each group. The experi- a higher variability (CV = 19.82% in the cortex of the VEH group). menter was blinded to treatment. SPM analysis nonetheless highlighted patterns of significantly higher glucose consumption in the THN102 group compared Determination of Modafinil Concentration in Serum with MOD alone (P < .05). The regional increase in MRGlu was and Brain consistent with the regional increase in the relative FDG uptake (Table  2; Figure  2). However, difference in MRGlu in the THN102 In parallel experiments, performed in another population of group compared with the VEH and FLE groups was not significant, rats, brain and serum concentrations of modafinil were deter - which may be due to a higher variability of the MRGlu data. mined after MOD and THN102 administration in rats. Animals (n = 8 rats/group) were anesthetized with isoflurane 30 minutes after i.v. administration and blood samples were collected, cen- THN102 Increases Spontaneous Locomotor Activity trifuged (3000 g× 15 min, 20°C), and stored at -80°C. Brains were To assess whether doses of modafinil allowed to discriminate collected immediately after blood sampling, freezed on dry ice, THN102 compared with modafinil alone, spontaneous motor and stored at -80°C. Modafinil and flecainide concentrations in activity in unrestrained awake rats was monitored for 2 hours after serum and brain lysate were determined using a reverse phase treatment, during the lights-on period (Figure 3). Locomotor activ- liquid chromatography with tandem mass spectrometry detec- ity in the MOD group tends to be increased compared with the tion technique (Eurofins ADME Bioanalyses). VEH group without reaching significance (+15.2% at 120 minutes). THN102 administration induced a significant increase in cumu- Statistical Analysis lative locomotor activity compared with VEH-treated animals, beginning at 95 minutes (+22.0% at 120 minutes vs VEH,P = .0309). Results of the locomotion and pharmacokinetic data are THN102 was more effective in increasing the locomotor activity expressed as mean ± SEM. Difference was considered signifi- compared with MOD; however, the difference was not statistically cant at P< .05 levels. Statistical analysis was performed using significant (+5.89% at 120 minutes). Moreover, no effect of FLE treat- Graphpad software (GraphPad Prism version 7). Cumulative ment was found at the 1-mg/kg dose when administered alone. traveled distance over time was compared using a 2-way repeated-measure ANOVA followed by Bonferroni’s posthoc test. Finally, serum and brain modafinil concentrations were com- Brain and Serum Concentrations of Modafinil pared using an unpaired t test. Serum and brain were collected 30 minutes after treatment with modafinil alone (MOD) or combined with flecainide (THN102; Results Figure  4). Quantification of modafinil concentration levels The goal of this study was to compare the brain metabolism treated with MOD or THN102 demonstrated no significant dif- after treatment with THN102, combination between modafinil ference between both groups in serum (2.12± 0.472  ng/mL and and flecainide at low dose, to modafinil alone. 2.14 ± 0.419 ng/mL, respectively) or in brain (1.37± 0.262 ng/g and Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 690 | International Journal of Neuropsychopharmacology, 2018 Table  1. SPM Results Showing Clusters, Peak Coordinates, and Significant Levels of Brain Regions with an Increase in F-FDG Uptake When Comparing THN102 to MOD, FLE, or VEH Significant Peak Coordinates within Clusters Intergroup Comparisons Significant Clusters x y z Peak Regions THN102 > VEH Cortex 0.5 4.6 3.6 R pre-limbic cortex Caudate and putamen (Striatum) 6.7 4.6 -5.6 R primary auditory cortex Amygdala -5.7 3 -8.8 L temporal association cortex -0.1 1.2 -0.2 L cingulate cortex area 1 -5.5 8.2 -0.6 L layer 3 of cortex THN102 > FLE Cortex 6.5 4.6 -5.8 R primary auditory cortex Caudate and putamen (Striatum) 4.3 2.8 2.0 R primary somatosensory Amygdala -5.5 8.6 -4.8 L layer 3 of cortex -2.1 3.6 3.8 L forceps minor corpus callosum -3.1 0.4 -7.8 L primary visual cortex monocular THN102 > MOD Cortex 5.7 7.2 -1.6 R dysgranular cortex Caudate and putamen (Striatum) 2.1 1.4 4.4 R secondary motor cortex Amygdala 2.5 0.8 -5.2 R secondary visual cortex -0.7 0.4 -4.2 L retrosplenialdysgranular cortex -3.7 6.8 3.0 L layer 3 of cortex -5.5 8.6 -4.8 L basolateral amygdaloïd nucleus, posterior Voxels comparison-based analysis was performed using SPM8 (n = 5) rats per group using 1-way ANOVA followed by multiple comparison test. A significance level threshold of .05 and a minimum cluster size of 200 voxels were selected to identify significant clusters. Coordinates of sig- nificant peak (P < .05) were given according to the Paxinos and Watson rat brain atlas (Paxinos and Watson, 2007). FLE, flecainide 1 mg/kg; MOD, modafinil 10 mg/kg; THN102, modafinil 10 mg/kg + flecainide 1 mg/kg; VEH, vehicle. 18 18 Figure 1. Coronal brain section showing statistical parametric map for increase in relativ F-2-fluor e o-2-deoxy-D-glucose ( F-FDG) uptake after Modafinil (MOD) + fle- cainide (THN102) treatment in comparison to MOD alone in rats. Following a cranio-caudal orientation centered on bregma (anterior is positive), coronal brain sections were at (a) 1.05, (b) -0.68, (c) -1.29, (d) -3.07, (e) -4.34, and (f) -5.76 millimeters from the bregma and (g) sagittal view of the brain with the marked coronal sections (red dotted lines) from a to f. Color scale represents all T distributions achieving statistical significance (see SPM statistical analysis). Acb, accumbens; Amy, amygdala; Cg, cingulate cortex; Ent, entorhinal cortex; Ic, insular cortex; M, motor cortex; RS, retrosplenial cortex; SS, somatosensory cortex; Str, striatum; V, visual cortex. 1.50 ± 0.277  ng/g, respectively). Therefore, there is no apparent Modafinil has been largely proposed in sleep medicine to pharmacokinetic interaction between MOD and FLE on MOD treat EDS associated with narcolepsy (Lavault et al., 2011 Thorp ; y metabolism. and Dauvilliers, 2015; Barateau et al., 2016), Parkinson’s disease (Sheng et  al., 2013; Rodrigues et  al., 2016), idiopathic hyper - somnia (Lavault et  al., 2011; Lopez et  al., 2017), and obstruct- Discussion ive sleep apnea/hypopnea syndrome (Black and Hirshkowitz, In this study, we reported that THN102 significantly activates 2005). Numerous preclinical studies have generated a wealth of experimental data, which lead to many hypotheses regard- the cortico-amygdala-striata regions compared with MOD used alone. ing the mode of action of modafinil (Gerrard and Malcolm, 2007; Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 Vodovar et al. | 691 Table 2. SPM Results Showing Clusters, Peak Coordinates, and Significant Levels of Brain Regions with an Increase in MRGlu When Comparing THN102 to MOD in Rats Significant Peak Coordinates within Clusters Intergroup Comparisons Significant Clusters x y z Peak Regions THN102 > MOD Cortex 4.1 0.8 -5.2 R primary visual cortex Caudate and putamen (Striatum) 0.7 2.2 3.8 R secondary motor cortex Amygdala 5.7 8.6 -4.6 R layer 3 of cortex Accumbens -1.5 0.6 -0.4 L primary motor cortex -4.9 9.0 0.0 L layer 3 of cortex -6.5 6.4 -8.8 L dorsolateral enthorinal cortex Voxels comparison based analysis was performed using SPM8 (n = 5) rats per group using 1-way ANOVA followed by multiple comparison test. A  significance level threshold of .05 and a minimum cluster size of 200 voxels were selected to identify significant clusters. Coordinates of significant peak (P < .05) were given according to the Paxinos and Watson rat brain atlas (Paxinos and Watson, 2007). MOD, modafinil 10 mg/kg; MRGlu, metabolic rate of glucose; THN102, modafinil 10 mg/kg + flecainide 1 mg/kg. Figure  2. Coronal brain section showing statistical parametric map for increase in absolute metabolic rate of glucose (MRGlu) after Modafinil (MOD) + flecainide (THN102) treatment in comparison to MOD in rats. Following a cranio-caudal orientation centered on bregma (anterior is positive), coronal brain sections were at (a) 1.05, (b) -0.68, (c) -1.29, (d) -3.07, (e) -4.34, and (f) -5.76 millimeters from the bregma; and (g) sagittal view of the brain with the marked coronal sections (red dotted lines) from a to f. Color scale represents all T distributions achieving statistical significance (see SPM statistical analysis). Acb, accumbens; Amy, amygdala; Cg, cingulate cor - tex; Ent, entorhinal cortex; Ic, insular cortex; M, motor cortex; RS, retrosplenial cortex; SS, somatosensory cortex; Str, striatum; V, visual cortex. Minzenberg and Carter, 2008). The central noradrenergic Recent data indicated that not only neurons but also glial cells, in hypothesis has been supported by data showing that inhibition particular astrocytes, were modulated by modafinil, as it enhanced of catecholamine synthesis or antagonism of adrenergic recep- astrocyte coupling and the expression of one of their connexins, tors is able to attenuate the wake-promoting effects of modafinil Cx30 (Liu et al., 2013). These data suggest that astroglial connexins (Duteil et  al., 1991; Lin et  al., 1992). Recent studies using mice might be involved in modafinil mode of action. Indeed, flecainide, lacking the noradrenaline synthesis or alpha1β-adrenoceptor an astroglial connexin inhibitor, was able to enhance modafinil (Stone et al., 2002a, 2002b; Hou et al., 2005) or brain imaging in procognitive and wake-promoting activities when coadministered humans (Minzenberg and Carter, 2008) also support the crit- to modafinil in rodents (Duchêne et al., 2016; Lu and Chen, 2016). ical involvement of the locus cœruleus noradrenergic system in THN102 is currently in phase II clinical trial on narcoleptic patients modafinil profile. The dopaminergic hypothesis has been very (NCT02821715). In this context, we compared the impact on brain attractive and prevailing since the identification of an affinity of functions between THN102 and modafinil in the rat by assessing modafinil with dopamine transporter (Mignot et al., 1994; Wisor their drug-induced changes in brain glucose metabolism. et al., 2001; Wisor and Eriksson, 2005; Korotkova et al., 2007Qu ; The route of administration and dose of modafinil et al., 2008). The brain disinhibitory hypothesis (Lin et al., 1996, (10 mg/kg, i.v.) was based on a previous functional magnetic res- 2000) has received less attention, yet it is supported by the fact onance imaging (fMRI) study in rats (Gozzi et al., 2012). Flecainide that modafinil induces a significant decrease in GABA outflow dose was chosen according to previous studies in rodents (Ferraro et al., 1996) in many brains areas, notably those critic- (Duchêne et al., 2016). Additionally, at selected doses, THN102 sig- ally involved in sleep-wake cycle control. nificantly increased locomotor activity, whereas modafinil alone Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 692 | International Journal of Neuropsychopharmacology, 2018 Figure 3. Cumulative traveled distance over 2 hours after vehicle (VEH), flecainide 1 mg/kg (FLE), modafinil 10 mg/kg (MOD), or MOD 10 mg/kg + FLE 1 mg/kg (THN102) treatment in awake rats. Locomotor activity of rats was measured during 2 hours after treatment. Data are expressed as means ± SEM (n = 8 rats/group) and compared using 2-way ANOVA followed by Bonferroni’s multiple comparison test: *P < .05 THN102 vs VEH. Figure 4. Serum (a) and brain (b) concentrations of modafinil after modafinil 10 mg/kg + flecainide 1 mg/kg (THN102) and modafinil 10 mg/kg (MOD) treatment. Thirty minutes after the treatment injection, serum and brain levels of MOD were sampled and quantified by liquid chromatography with tandem mass spectrometry. Data are expressed as means with scattered plots ± SEM (n = 8 rats/group) and compared using an unpaired t test. showed only a tendency to increase it (nonsignificant), as partially Gozzi et  al., 2012), including fMRI, 2-deoxyglucose autoradi- described elsewhere (Simon et  al., 1996; Edgar and Seidel, 1997; ography, FDG PET, and cerebral blood flow assesement using Zolkowska et al., 2009). As previously reported in mice (Duchêne single-photon emission computed tomography. These data et al., 2016), assessment of modafinil concentration in the serum tend towards a consensus over cortical and subcortical brain and brain confirmed that flecainide did not increase the brain activations induced by modafinil including hypothalamic and distribution of modafinil. Therefore, pharmacokinetic interaction thalamic regions (Thomas and Kwong, 2006; Gozzi et al., 2012), between flecainide and modafinil can unlikely explain the brain which are highly involved in the regulation of sleep-wake cycle effect of THN102 compared with vehicle and modafinil alone. (Szabadi, 2006; Lin et al., 2011). Other major structures are high- Serum concentrations were close to modafinil levels found in lighted following modafinil administration as the caudate puta- subjects following dosing of clinically effective doses (McClellan men (striatum), the amygdala, and the hippocampus (Engber and Spencer, 1998; Wong et  al., 1999), suggesting potential rele- et al., 1998; Ghahremani et al., 2011), known for their implication vance of the presented data to clinical conditions. in driving emotions and cognitive functions (Joo et al., 2008a). Several methods have been proposed to investigate the CNS In our conditions, modafinil alone did not modulate glucose effects of modafinil in human (Ellis et  al., 1999; Spence et  al., brain metabolism assessed by FDG PET compared with vehicle. 2005; Hunter et  al., 2006; Thomas and Kwong, 2006; Kim et  al., Even though the dosage and the route were identical to a previ- 2007; Joo et al., 2008a; Minzenberg et al., 2008; Rasetti et al., 2010; ous fMRI study (Gozzi et  al., 2012), the observed cortical acti- Ghahremani et  al., 2011; Minzenberg et  al., 2011; Goudriaan vation after treatment was not detected in our analysis. This et al., 2013; Schmaal et al., 2013; Funayama et al., 2014; Schmaal difference highlights the discrepancies that may exist between et al., 2014; Ikeda et al., 2017; Schmidt et al., 2017) and animals the drug-induced response on blood oxygenation levels- (Engber et  al., 1998; van Vliet et  al., 2008; Dawson et  al., 2012; dependent response and on energy consumption (Di et al., 2012; Downloaded from https://academic.oup.com/ijnp/article-abstract/21/7/687/4938109 by Ed 'DeepDyve' Gillespie user on 03 July 2018 Vodovar et al. | 693 Cabrera et  al., 2016). Another explanation for such discrepan- sleep-wake cycle and behaviors. Our results further support cies may be the presence of anesthesia. In the present study, the hypothesis that astrocyte connexins are involved in phar - animals were exposed to <2.5% isoflurane during drug admin- macological responses of psychoactive drugs such as modafinil istration and subsequent PET acquisition. It was reported that (Duchêne et al., 2016; Jeanson et al., 2016; Charvériat et al., 2017). isoflurane may modulate the brain glucose uptake and may thus limit the sensitivity of the method to detect the CNS response Funding to investigated compounds in vivo (Spangler-Bickell et al., 2016; Park et al., 2017). Working on awake rodents was shown feasible, This work was supported by Theranexus Company and the although technically challenging (Spangler-Bickell et  al., 2016; Agence Nationale de la Recherche (grant number 14-CE16-0022). Park et  al., 2017). In the present study, we chose to administer investigated treatments under isoflurane anesthesia to facilitate Acknowledgments animal handling, i.v. administration, and avoid any stress due to the experimental procedure. Moreover, we showed that the loco- We thank Emile Jaumain for helpful technical assistance. motor activity was different between groups, which may non- specifically impact FDG uptake by the brain in awake animals. Statement of Interest Therefore, the whole procedure was performed under isoflurane anesthesia to highlight the intrinsic effect of each treatment on F.M., M.C., and A.D. are full-time employees of Theranexus com- brain function and allow for dynamic PET acquisition for 60 pany. Y.D.  served as consultant for Bioprojet Pharma, Flamel minutes in immobile animals. Technologies, Jazz Pharmaceuticals, Theranexus, Takeda, and Some brain structures such as the locus coeruleus, thala- UCB. The other authors declare no financial conflict of interest. mus, and hypothalamus, modulated by modafinil (Minzenberg et  al., 2008; Gozzi et  al., 2012; Schmaal et  al., 2013) were not References activated by THN102 compared with modafinil alone. More Bao Q, Newport D, Chen M, Stout DB, Chatziioannou AF (2009) interestingly, this study reported that THN102 significantly Performance evaluation of the inveon dedicated PET preclin- increased the relative glucose brain uptake in the whole cortex, ical tomograph based on the NEMA NU-4 standards. J Nucl amygdala, and striatum compared with modafinil alone, hence Med 50:401–408. suggesting a different activity on glucose metabolism between Barateau L, Lopez R, Dauvilliers Y (2016) Treatment options for both treatment groups. Similar effects could be observed using narcolepsy. CNS Drugs 30:369–379. pharmacokinetic modelling and estimation of the absolute Bayard S, Croisier Langenier M, Cochen De Cock V, Scholz S, MRGlu. Despite higher variability in this parameter, patterns of Dauvilliers Y (2012) Executive control of attention in narco- increased metabolic rate of glucose were detected in the cortex, lepsy. Plos One 7:e33525. striatum, amygdala, and accumbens of rats in the THN102 group Béracochéa D, Celerier A, Peres M, Pierard C (2003) Enhancement compared with the MOD group. Such brain regions may be con- of learning processes following an acute modafinil injection sidered as substrates for the enhancement of modafinil effects in mice. Pharmacol Biochem Behav 76:473–479. by flecainide. They notably process attention states and motiva- Black JE, Hirshkowitz M (2005) Modafinil for treatment of tion and code behavioral responses (Cardinal et  al., 2002 Cho ; residual excessive sleepiness in nasal continuous positive et al., 2013), functions that are altered in narcolepsy and that are airway pressure-treated obstructive sleep apnea/hypopnea portentially modulated by THN102 (Bayard et al., 2012). syndrome. Sleep 28:464–471. 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Neurosci Biobehav Rev 26:321–352. demonstrated at least in the amygdala (Johnson et al., 2010) and Charvériat M, Naus CC, Leybaert L, Sáez JC, Giaume C (2017) cortex (Sims et  al., 2015), providing potential evidence for the Connexin-dependent neuroglial networking as a new thera- involvement of astrocyte networks in modafinil pharmacologi- peutic target. Front Cell Neurosci 11:174. cal profile (Duchêne et al., 2016). Cho YT, Ernst M, Fudge JL (2013) Cortico-amygdala-striatal cir - Furthermore, our data confirm, for the first time, the greater cuits are organized as hierarchical subsystems through the effects of THN102 compared with modafinil in terms of brain primate amygdala. 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International Journal of NeuropsychopharmacologyOxford University Press

Published: Mar 15, 2018

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