0022-3565/09/3292-738–746 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 329, No. 2 U.S. Government work not protected by U.S. copyright 146142/3459720 JPET 329:738–746, 2009 Printed in U.S.A. Evidence for the Involvement of Dopamine Transporters in Behavioral Stimulant Effects of Modafinil Dorota Zolkowska, Raka Jain, Richard B. Rothman, John S. Partilla, Bryan L. Roth, Vincent Setola, Thomas E. Prisinzano, and Michael H. Baumann Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland (D.Z., R.B.R., J.S.P., M.H.B.); Department of Hygiene, Medical University of Lublin, Lublin, Poland (D.Z.); National Drug Dependence Centre, All India Institute of Medical Sciences, Ansari Nagar, New Dehli, India (R.J.); Department of Pharmacology, Medicinal Chemistry, and Psychiatry, University of North Carolina School of Medicine, Chapel Hill, North Carolina (B.L.R.,V.S.); and Department of Medicinal Chemistry, The University of Kansas, Lawrence, Kansas (T.E.P.) Received September 15, 2008; accepted January 26, 2009 ABSTRACT Modafinil is prescribed for numerous medical conditions, but DAT substrate [3H]1-methyl-4-phenylpyridinium. Intravenous the drug’s mechanism of action is unclear. Here, we examined modafinil (20 and 60 mg/kg) produced dose-dependent in- the interaction of modafinil with receptors and transporters in creases in motor activity and extracellular DA, without affect- vitro and compared pharmacological effects of the drug with ing serotonin (5-HT). Analogous results were observed for those produced by indirect dopamine (DA) agonists 1-[2-[bis(4- GBR12909 (1 and 3 mg/kg), whereas METH (0.3 and 1 mg/ fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine kg) increased DA and 5-HT. Locomotor effects of all drugs (GBR12909) and (ϩ)-methamphetamine (METH). Modafinil was were positively correlated with dialysate DA (P Ͻ 0.001). screened at various receptors and transporters using binding Interestingly, modafinil pretreatment reduced METH-induced assays. Transporter-mediated uptake and release were exam- ambulation and DA release. Our data show that modafinil ined in rat brain synaptosomes. Effects of modafinil on motor interacts with DAT sites in rat brain, a property shared with activity and neurochemistry were determined in rats undergo- agonist medications under investigation for treating cocaine ing in vivo microdialysis in nucleus accumbens. Of the recep- dependence. Nondopaminergic mechanisms may also con- tors and transporters assayed, modafinil displayed measurable tribute to the pharmacology of modafinil. Finally, the results potency only at DA transporters (DAT), inhibiting [3H]DA uptake, suggest that modafinil should be tested as an adjunct for ␮ with an IC50 value of 4.0 M. Accordingly, modafinil pre- treating METH addiction. treatment (10 ␮M) antagonized METH-induced release of the Modafinil (2-[(diphenylmethyl) sulfinyl] acetamide) is a This research was supported in part by the Intramural Research wake-promoting agent approved for the treatment of narco- Program of the National Institutes of Health National Institute on Drug lepsy (Wise et al., 2007). Recently, modafinil has been pre- Abuse. scribed for other psychiatric disorders such as attention- R.J. is supported by the National Institutes of Health National Institute on Drug Abuse Distinguished International Scientist Collaboration Award deficit hyperactivity disorder (Swanson et al., 2006) and Program. cocaine dependence (Dackis et al., 2005). In a clinical labo- This work was presented previously. Baumann MH, Zolkowska D, Jain R, Partilla JS, Prisinzano TE, and Rothman RB (2008) Evidence that ratory setting, modafinil pretreatment reduces cocaine self- stimulant effects of modafinil in rats involve dopamine transporters. Col- administration (Hart et al., 2008) and positive subjective lege on Problems of Drug Dependence, 70th Annual Scientific Meeting; 2008 June 18; San Juan, P.R. College on Problems of Drug Dependence, Phila- effects (Dackis et al., 2003; Malcolm et al., 2006), supporting delphia, PA. the utility of the drug as a pharmacotherapy for stimulant Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. addiction. The off-label use of modafinil for treating cocaine doi:10.1124/jpet.108.146142. dependence is noteworthy because no approved medications ABBREVIATIONS: DA, dopamine; 5-HT, 5-hydroxytryptamine (serotonin); DAT, dopamine transporter(s); NE, norepinephrine; NET, norepineph- rine transporter(s); GBR12935, 1-[2-benzhydroxyethyl]-4-(3-phenylpropyl)piperazine; METH, (ϩ)-methamphetamine; RTI-55, 3␤-(4-iodophenyl)- tropan-2␤-carboxylic acid methyl ester; SERT, serotonin transporter(s); MPPϩ, 1-methyl-4-phenylpyridinium; HPLC-ECD, high-pressure liquid chromatography with electrochemical detection; ANOVA, analysis of variance; MeNER, (S,S)-2-(␣-(2-methoxyphenoxy)benzyl)morpholine; n., nucleus; SCH23390, R-(ϩ)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; GR125743, N-[4-methoxy-3-(4-methyl piperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide; MK-801, 5H-dibenzo[a,d]cyclohepten-5,10-imine (dizocilpine maleate); [125I]HEAT, [125I]iodo-2-[␤-(4-hydroxyphenyl)-ethylaminomethyl]tetralone; [3H]N-methylspiperone. 738 Modafinil and Dopamine Transporters 739 are available for this disease. Despite the widespread clinical rats were anesthetized with sodium pentobarbital (60 mg/kg i.p.), use of modafinil, the mechanisms underlying its therapeutic and then jugular catheters and intracerebral guide cannulae were efficacy are not well understood (for review, see Ballon and implanted (Baumann et al., 2002). Guide cannulae were aimed at the Feifel, 2006). n. accumbens according to coordinates ML, Ϫ1.7 mm and AP, ϩ1.6 Ϫ Initial studies in animals demonstrated that stimulant mm relative to bregma, and DV, 6.0 mm relative to dura. Rats were single-housed postoperatively and allowed at least 1 week to recover. effects of modafinil are distinct from those of amphetamine In Vitro Receptor and Transporter Assays. Binding and func- and may not involve dopamine (DA) systems in the brain tional assays were carried out in transfected cells expressing cloned (Duteil et al., 1990; Simon et al., 1995). The reported nondo- human G protein-coupled receptors or monoamine transporters, us- paminergic effects of modafinil include activation of ␣1 ad- ing resources of the National Institute of Mental Health Psychoac- renergic receptors (Duteil et al., 1990), enhancement of sero- tive Drug Screening Program (http://pdsp.med.unc.edu/pdspw/ tonin (5-HT) function (Ferraro et al., 2000), inhibition of clones.php). Multiple receptor subtypes for DA, NE, 5-HT, GABA, GABA release (Ferraro et al., 1997, 1998), and stimulation of glutamate, histamine, and opioids were investigated, and details of glutamate and histamine release (Ferraro et al., 1999; Ishi- assay conditions have been published previously (Roth et al., 2002). zuka et al., 2003). With regard to the treatment of cocaine Binding assays for monoamine transporters were also carried out in rat brain membranes; the cocaine analog [125I]RTI-55 was used to dependence, modafinil has been described as a glutamate 3 enhancer (Dackis and O’Brien, 2003). In contrast, substan- label DAT and 5-HT transporters (SERT), whereas [ H]nisoxetine was used to label NET. The initial screening of modafinil at receptors tial evidence indicates that modafinil may exert its effects via and transporters was performed in quadruplicate at a 10 ␮M con- presynaptic dopaminergic mechanisms (Nishino et al., 1998; centration. In instances where modafinil displayed greater than 50% Minzenberg and Carter, 2008). Mignot et al. (1994) reported inhibition of binding, Ki values were determined using six to 10 in 1994 that modafinil inhibits DA transporter (DAT) bind- concentrations of the drug. Uptake and release assays for DAT, NET, ␮ ing, with an IC50 value of 3.2 M (Mignot et al., 1994), and SERT were carried out in rat brain synaptosomes using pub- whereas Madras et al. (2006) showed recently that modafinil lished methods (Rothman et al., 2003). For uptake inhibition assays, occupies DAT and norepinephrine (NE) transporters (NET) [3H]DA, [3H]NE, and [3H]5-HT were used to assess transport activity 3 in living primate brain. Consistent with these data, modafi- at DAT, NET, and SERT, respectively. In release assays, [ H]1- methyl-4-phenylpyridinium ([3H]MPPϩ) was used as a radiolabeled nil administration increases extracellular levels of DA in 3 brain as measured by in vivo microdialysis (de Saint Hilaire substrate for DAT and NET, whereas [ H]5-HT was used as a sub- strate for SERT. K values for receptor binding, IC values for et al., 2001; Wisor et al., 2001; Murillo-Rodríguez et al., i 50 uptake inhibition, and Ke values (i.e., apparent dissociation constant 2007), and wake-promoting actions are absent in DAT-knock- of an antagonist) for METH-induced release were calculated using out mice (Wisor et al., 2001). GraphPad Prism version 4.0 (GraphPad Software Inc., San Diego, Based on available evidence, it seems that modafinil inter- CA). acts with multiple molecular targets in the brain, including In Vivo Microdialysis and Motor Activity. On the evening DAT proteins. Nonetheless, there are fundamental unre- before microdialysis testing, rats were brought into the laboratory. solved issues regarding the pharmacology of modafinil. For Extension tubes were connected to catheters, and microdialysis