Author Manuscript NIH Public Access for Stimulant Addiction ......1997). For instance, acute or...

Dual Dopamine/Serotonin Releasers: Potential Treatment Agentsfor Stimulant Addiction

Richard B. Rothman,National Institute on Drug Abuse, National Institutes of Health

Bruce E. Blough, andResearch Triangle Institute International

Michael H. BaumannNational Institute on Drug Abuse, National Institutes of Health

Abstract“Agonist therapy” for cocaine and methamphetamine addiction involves administration of stimulant-like medications (e.g., monoamine releasers) to reduce withdrawal symptoms and prevent relapse.A significant problem with this strategy is that many candidate medications possess abuse liabilitybecause of activation of mesolimbic dopamine (DA) neurons in the brain. One way to reduce DA-mediated abuse liability of candidate drugs is to add in serotonin (5-HT) releasing properties, sincesubstantial evidence shows that 5-HT neurons provide an inhibitory influence over mesolimbic DAneurons. This article addresses several key issues related to the development of dual DA/5-HTreleasers for the treatment of substance use disorders. First, the authors briefly summarize theevidence supporting a dual deficit in DA and 5-HT function during withdrawal from chronic cocaineor alcohol abuse. Second, the authors discuss data demonstrating that 5HT release can dampen DA-mediated stimulant effects, and the “antistimulant” role of 5-HT2C receptors is considered. Next, themechanisms underlying potential adverse effects of 5-HT releasers are described. Finally, the authorsdiscuss recently published data with PAL-287, a novel nonamphetamine DA/5-HT releasing agentthat suppresses cocaine self-administration but lacks positive reinforcing properties. It is concludedthat DA/5-HT releasers could be useful therapeutic adjuncts for the treatment of cocaine and alcoholaddictions, as well as for obesity, attention-deficit disorder, and depression.

Keywordsalcohol; amphetamine; cocaine; dopamine; serotonin; transporter

This article reviews data from our laboratory pertaining to the development of dual dopamine(DA)/serotonin (5-HT) releasers as medications for stimulant addiction, and possibly alcoholaddiction (Baumann et al., 2000; Baumann, Ayestas, Dersch, & Rothman, 2001; Rea, Rothman,& Shippenberg, 1998; Rothman & Baumann, 2003; Rothman et al., 2005; Wee et al., 2005;Wojnicki, Rothman, Rice, & Glowa, 1999). We also integrate our findings with existingliterature to provide a conceptual framework for the design of new medications for addictiondisorders. The term “stimulant” refers to drugs like cocaine and amphetamines that produce a

Correspondence concerning this article should be addressed to Richard B. Rothman Clinical Psychopharmacology Section, IRP/NIDA/NIH, Clinical Psychopharmacology Section, Suite 4500, Triad Building, 333 Cassell Drive, Baltimore, MD 21224. E-mail: E-mail:[email protected] B. Rothman and Michael H. Baumann, Clinical Psychopharmacology Section, Intramural Research Program, National Instituteon Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland; Bruce E. Blough,Chemistry and Life Sciences Group, Research Triangle Institute International, Research Triangle Park, North Carolina.

NIH Public AccessAuthor ManuscriptExp Clin Psychopharmacol. Author manuscript; available in PMC 2009 December 1.

Published in final edited form as:Exp Clin Psychopharmacol. 2008 December ; 16(6): 458–474. doi:10.1037/a0014103.

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spectrum of effects in humans including mood elevation, cardiovascular stimulation, and adecreased need for sleep. At high doses, or after longer periods of use, stimulants can cause arange of adverse effects, such as disordered thoughts and psychotic episodes. In laboratoryanimals, stimulants increase locomotor activity and are readily self-administered because oftheir powerful reinforcing properties. Figure 1 shows the chemical structures of drugs discussedin this article. Many of these drugs are useful medications with long histories of efficacy andsafety. Others are highly addictive substances associated with considerable morbidity andmortality (Anonymous, 1995; Das, 1993; Gonzalez Castro, Barrington, Walton, & Rawson,2000; Musto, 1992). In some cases, as with amphetamine, the same drug can be a therapeuticentity or an abused substance, depending upon the context in which the drug is administered(Arnsten, 2006; Greenhill, 2006).

Most stimulant drugs interact with monoamine neurons in the central nervous system (CNS).Neurons that synthesize, store, and release monoamine transmitters—norepinephrine (NE),DA, and 5-HT—are widely distributed in the mammalian CNS. These neurons expressspecialized plasma membrane proteins, which function to transport previously releasedtransmitter molecules from the extracellular space back into the cytoplasm (Amara & Kuhar,1993; Masson, Sagne, Hamon, & el Mestikawy, 1999). It is well established that distincttransporter proteins are expressed by each type of monoamine neuron: NE transporters (NETs),DA transporters (DATs), and 5-HT transporters (SERTs) are associated with NE, DA, and 5-HT neurons, respectively. These proteins belong to a superfamily of Na+/Cl−-dependenttransporters that share genetic, morphological, and functional homologies (Torres & Amara,2007; Uhl & Johnson, 1994). Under normal circumstances, the transporter-mediated uptake ofmonoamine transmitters is the principal mechanism for inactivation of monoamine signalingin the brain. Consequently, drugs that interact with monoamine transporters have profoundeffects on CNS function, and these effects can be beneficial or detrimental depending upon thedose, route, and formulation of the drug administered (Amara & Sonders, 1998; Iversen,2006).

Drugs that target transporter proteins can be divided into two classes based on their precisemechanism of action—reuptake inhibitors and substrate-type releasers (Rothman & Baumann,2003). Reuptake inhibitors bind to transporter proteins but are not transported. These drugselevate extracellular transmitter concentrations by blocking transporter-mediated recapture oftransmitter molecules from the synapse. Substrate-type releasers bind to transporter proteinsand are subsequently transported into the cytoplasm of nerve terminals. Thus, transmitter“releasers” are often referred to as transporter “substrates.” Releasers elevate extracellulartransmitter concentrations by a two-pronged mechanism: (1) they promote efflux of transmitterby a process of transporter-mediated exchange, and (2) they increase cytoplasmic levels oftransmitter by disrupting storage of transmitters in vesicles via interactions with the vesicularmonoamine transporter 2 (Fleckenstein, Volz, Riddle, Gibb, & Hanson, 2007; Rudnick,1997). The molecular mechanisms underlying transporter-mediated transmitter release are notcompletely understood, but ionic currents, oligomerization, and reversal of normal transportfunction appear to be involved (Blakely, Defelice, & Galli, 2005; Sitte & Freissmuth, 2003;Sulzer, Sonders, Poulsen, & Galli, 2005). Because substrate-type releasing agents must betransported into nerve terminals to promote transmitter release, reuptake inhibitors can blockthe effects of releasers.

A Dual Deficit Model of Stimulant AddictionThe use of stimulants such as cocaine and methamphetamine produces a “high” or “rush” thatis likely mediated by elevations in extracellular DA levels in mesolimbic circuits (Di Chiaraet al., 2004; Volkow, Fowler, & Wang, 2002), although some evidence indicates that elevationsin extracellular NE may also contribute (Alexander et al., 2005; Rothman et al., 2001).

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Similarly, alcohol-induced increases in extracellular DA are thought to underlie the positivereinforcing effects of this commonly abused substance (Koob, 2003; Koob et al., 1998).Repeated misuse of stimulants, especially when they are self-administered via the smoked orintravenous routes, can lead to serious addiction in susceptible individuals. The chronic abuseof stimulants and alcohol, despite negative consequences, causes long-term changes inneurochemistry, including changes in the γ-aminobutyric acid and glutamate systems, and braincircuitry via processes of synaptic plasticity (Hyman, 2005; Kalivas & O’Brien, 2008; Volkow& Li, 2004).

Preclinical and human research findings demonstrate that withdrawal from stimulant andalcohol abuse is associated with deficits in DA and 5-HT function. For example, rats withdrawnfrom chronic cocaine or alcohol administration display decreased levels of extracellular DAand 5-HT in the nucleus accumbens (Parsons, Koob, & Weiss, 1995; Parsons, Smith, & Justice,1991; Rossetti, Hmaidan, & Gessa, 1992; Weiss et al., 1996). Human brain imaging studiesshow that cocaine addicts have reductions in evoked DA release and a reduction in DA D2-binding potential in the striatum (Martinez et al., 2007; Volkow et al., 2002, 1997).Neuroendocrine responsiveness to 5-HT releasers is diminished in rats withdrawn fromrepeated cocaine injections (Baumann, Becketts, & Rothman, 1995; Levy, Li, & Van de Kar,1994), and similar findings have been reported in abstinent human cocaine addicts (Ghitza,Rothman, Gorelick, Henningfield, & Baumann, 2007; Haney, Ward, Gerra, & Foltin, 2001).Taken together, these data suggest a cardinal feature of withdrawal from chronic cocaine, andpossibly alcohol, is decreased synaptic levels of DA and 5-HT in critical brain circuits.

Additional clinical support for the existence of 5-HT deficits in cocaine addicts is theoccurrence of symptoms resembling major depression during abstinence (Dackis & Gold,1985; Gawin & Kleber, 1986), coupled with an increased prevalence of suicidal ideation andsuicide attempts (Garlow, Purselle, & D’Orio, 2003). The well-accepted importance of 5-HTdysfunction in mediating depression and suicide (for review, see Mann, 2003) suggests aparallel role for decreased synaptic 5-HT in cocaine and alcohol withdrawal states (Lesch,2005). Indeed, the spectrum of symptoms often reported by patients withdrawing fromstimulant or alcohol use—suicidal ideations, depressed mood, obsessive thoughts, intensecraving, anhedonia, increased impulsivity, and susceptibility to drug-related cues—presumably reflects long-term changes in brain function and structure. We have proposed thatdeficits in monoamine systems underlie at least some of the symptoms experienced duringwithdrawal (for review, see Baumann & Rothman, 1998b).

In particular, we proposed a dual deficit model of stimulant addiction in which drug-inducedDA and 5-HT dysfunction contributes to withdrawal symptoms, drug craving, and relapse(Baumann, 2000; Baumann & Rothman, 1998a, 1998b; Rothman, Elmer, Shippenberg, Rea,& Baumann, 1998). Depicted diagrammatically in Figure 2, the dual deficit model postulatesthat decreased synaptic DA during stimulant withdrawal underlies anhedonia and psychomotorretardation, whereas decreased synaptic 5-HT gives rise to depressed mood, obsessivethoughts, and lack of impulse control. Consistent with this model, rats receiving repeatedinjections of abused stimulants exhibit neurobiological changes similar to those observed inhuman patients with major depression (Baumann, Becketts et al., 1995; Baumann & Rothman,1998a; Lin, Koob, & Markou, 1999; Markou & Koob, 1991). If abstinent stimulant addictsexhibit DA and 5-HT deficits, medications capable of correcting abnormalities in DA and 5-HT function might be effective in treating stimulant and alcohol dependence.

In agreement with the dual deficit hypothesis, drugs that release DA (phentermine,amphetamine) or 5-HT (fenfluramine) display properties consistent with the effectivetreatment of substance use disorders (Halladay, Wagner, Hsu, Sekowski, & Fisher, 1999;Rothman et al., 1998; Rothman, Gendron, & Hitzig, 1994; Yu, Fisher, Sekowski, & Wagner,



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1997). For instance, acute or chronic administration of low doses of DA releasers, such as d-amphetamine, decreases cocaine self-administration behavior in rhesus monkeys (Macacamulatta; Glowa, Wojnicki, Matecka, Rice, & Rothman, 1995; Negus & Mello, 2003a,2003b). The data in Figure 3 demonstrate that the DA releasing agent phentermine suppressesresponding for cocaine injections without affecting food-reinforced behavior, and this effectis maintained by daily administration of phentermine (Wojnicki et al., 1999). Such preclinicalresults provide a rationale for using DA releasers as medications for treating cocaine addiction(Grabowski, Shearer, Merrill, & Negus, 2004; Lile, 2006; Rothman, Blough, & Baumann,2002).

Under certain conditions, the 5-HT releaser fenfluramine decreases responding for cocaine inrhesus monkeys as well (Negus, Mello, Blough, Baumann, & Rothman, 2007a). Combinedadministration of phentermine plus fenfluramine produces a 75% decrease in cocaine self-administration in monkeys (Glowa, Rice, Matecka, & Rothman, 1997). The mixture ofphentermine and d-fenfluramine reduces cocaine self-administration by 80% in rats, yet thismixture is not self-administered (Glatz et al., 2002). Important to note, 5-HT releasing agentssuppress cue-elicited cocaine-seeking behavior in rats (Burmeister, Lungren, & Neisewander,2003) and decrease cocaine craving in cocaine-dependent human patients (Buydens-Brancheyet al., 1998). The collective findings suggest that combined treatment with DA and 5-HTreleasers may have greater therapeutic value than treatment with either drug alone, in terms ofdecreasing stimulant self-administration and reducing cue-induced relapse. Moreover, agrowing body of evidence shows that DA/5-HT-releasing agents may provide similartherapeutic benefits for alcohol dependence (Halladay et al., 1999; Halladay et al., 2006; Yuet al., 1997).

5-HT Release Counteracts Stimulant Effects of DA ReleaseThe use of stimulant-like medications to treat stimulant addictions is an approach known as“agonist” therapy. This strategy involves administering medications that are less potent andless addictive than cocaine or methamphetamine, but which decrease stimulant abuse becauseof shared neurochemical properties with the abused drugs (Gorelick, 1998). Accordingly, wehave described agonist therapy as neurochemical “normalization” therapy—that is, thestimulant medication serves to normalize or stabilize neurochemical deficits caused by chronicexposure to the abused stimulant (Rothman & Baumann, 2003; Rothman et al., 2002).Neurochemical normalization therapy has generated effective treatments for nicotinedependence (Henningfield, 1995; Rollema et al., 2007) and opioid dependence (Ling, Rawson,& Compton, 1994; White & Lopatko, 2007). This approach has been explored for the treatmentof cocaine dependence as well (Alim, Rosse, Vocci, Lindquist, & Deutsch, 1995; Grabowskiet al., 1997; Kampman et al., 2000; Walsh, Haberny, & Bigelow, 2000), and a number ofplacebo-controlled trials have shown promising results (Grabowski et al., 2001; Grabowski,Rhoades et al., 2004; Shearer, Wodak, van Beek, Mattick, & Lewis, 2003). A significantlimitation of this strategy, however, is that candidate medications often exhibit inherent abuseliability due to activation of mesolimbic DA neurons in the brain (for review, see Grabowski,Shearer et al., 2004).

One feasible means to decrease the abuse liability of candidate medications is to add 5-HT-releasing properties to these drugs. Several lines of evidence support the hypothesis thatelevations in synaptic 5-HT counteract the stimulant and reinforcing effects mediated byelevations in synaptic DA (Burmeister, Lungren, Kirschner, & Neisewander, 2004; Czoty,Ginsburg, & Howell, 2002; Daw, Kakade, & Dayan, 2002; Higgins & Fletcher, 2003).Administration of the 5-HT precursor L-tryptophan, which increases 5-HT synthesis andrelease in the CNS, decreases self-administration of cocaine and amphetamine in rats(McGregor, Lacosta, & Roberts, 1993; Smith, Yu, Smith, Leccese, & Lyness, 1986). Likewise,



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pretreatment with 5-HT reuptake inhibitors can reduce intravenous cocaine self-administrationin rats and squirrel monkeys (Carroll, Lac, Asencio, & Kragh, 1990; Howell & Byrd, 1995).Cocaine analogs that have potent affinity at SERT support less self-administration behaviorthan analogs with weak affinity for SERT (Howell, Carroll, Votaw, Goodman, & Kimmel,2007; Roberts et al., 1999). Consistent with these findings, agents that broadly activate brain5-HT systems can reduce self-administration of stimulants and other drugs of abuse (Higgins& Fletcher, 2003). The “antistimulant” effects of increasing extracellular 5-HT are readilyobserved after combined administration of 5-HT releasers and DA releasers, or afteradministration of single agents that release both neurotransmitters.

As summarized in Table 1, drugs that release [3H]DA more potently than [3H]5-HT in vitro(e.g., amphetamine, phentermine) increase endogenous extracellular DA more thanextracellular 5-HT in vivo. Such indirect DA agonists are strong locomotor stimulants andsupport self-administration behavior. Drugs that release [3H]5-HT more potently than [3H]DAin vitro (e.g., fenfluramine, chlorphentermine) increase endogenous extracellular 5-HT morethan extracellular DA. Such indirect 5-HT agonists produce minimal motor activity and do notsupport self-administration behavior. The antireward effect of 5-HT releasers is also seen inthe conditioned place preference (CPP) assay as shown in Figure 4, where a low dose offenfluramine greatly reduces the positive CPP induced by phentermine. Consistent with thesefindings, fenfluramine administration decreased the subjective effects of D-amphetamine orphentermine in humans (Brauer, Johanson, Schuster, Rothman, & de Wit, 1996).

It seems possible that attempts to reduce the abuse liability of a DA releaser via elevation ofextracellular 5-HT could reduce therapeutic effectiveness. Some recent preclinical data supportthis contention (Negus, Mello, Blough, Baumann, & Rothman, 2007b). In particular, Negusand coworkers demonstrated that pretreatment of rhesus monkeys (Macaca mulatta) withselective DA releasers can suppress cocaine self-administration without affecting food-maintained responding. By contrast, mixed DA/5-HT releasers suppressed both cocaine- andfood-maintained responding under identical test conditions. These data suggest that selectiveDA releasers might be more effective than mixed releasers in the treatment of cocainedependence, but this hypothesis can only be addressed by carefully controlled clinical trials inhuman subjects.

The exact mechanisms responsible for antistimulant effects of 5-HT releasers have not beencharacterized, but increases in extracellular 5-HT would be expected to activate multiple 5-HTreceptor subtypes known to modulate DA function (Alex & Pehek, 2007; Muller & Huston,2006). As mentioned previously, the stimulant and reinforcing properties of drugs like cocaineand methamphetamine are mediated via activation of mesolimbic DA neurons. Cell bodies ofmesolimbic DA neurons reside in the midbrain ventral tegmental area (VTA) and send axonalprojections to many regions of the forebrain, most notably the nucleus accumbens (Moore &Bloom, 1978; Ungerstedt, 1971). The nucleus accumbens is a critical limbic-motor interfacereceiving afferent inputs from the prefrontal cortex, hippocampus and amygdala, while sendingefferent outputs to the ventral pallidum and other regions known to modulate locomotor centersin the brainstem (Mogenson, Jones, & Yim, 1980; Pennartz, Groenewegen, & Lopes da Silva,1994). 5-HT neurons in the midbrain raphe nuclei send axonal projections that denselyinnervate the mesolimbic system at the level of the VTA and nucleus accumbens (Molliver,1987; Steinbusch, 1981). Furthermore, 5-HT neurons innervate various regions providingexcitatory afferents to the accumbens (e.g., prefrontal cortex). In this manner, 5-HT nerveterminals are well positioned to influence the activity of mesolimbic DA neurons at multiplelevels.

The serotonergic modulation of DA function is inherently complex due to the presence of atleast 14 different 5-HT receptor subtypes in the CNS (Barnes & Sharp, 1999; Hoyer, Hannon,



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& Martin, 2002). While some 5-HT receptor subtypes enhance DA transmission (Alex &Pehek, 2007; Muller & Huston, 2006), 5-HT2C receptors provide a strong inhibitory influenceon mesolimbic DA neurons (Bubar & Cunningham, 2006; Di Matteo, De Blasi, Di Giulio, &Esposito, 2001). For instance, systemic administration of the 5-HT2C agonist RO60-0175markedly inhibits DA cell firing in the VTA and decreases extracellular levels of DA inforebrain projection areas (Di Matteo, Di Giovanni, Di Mascio, & Esposito, 1999, 2000; Gobertet al., 2000). Pretreatment with RO60-0175 reduces locomotor activity and self-administrationbehavior produced by cocaine, whereas pretreatment with the 5-HT2C antagonist SB242,084has the opposite effect (Fletcher, Grottick, & Higgins, 2002; Grottick, Fletcher, & Higgins,2000). In fact, SB242,084 given alone increases burst firing of DA cells in the VTA, suggesting5-HT2C receptors provide tonic inhibition of mesolimbic DA activity. Collectively, thesefindings indicate a potential role for 5-HT2C receptors in mediating the antistimulant effectsof 5-HT releasers (Higgins & Fletcher, 2003).

Recent data show that antistimulant effects of 5-HT2C receptor activation involve at least twoseparate mechanisms—one mechanism in the VTA and another in prefrontal cortex.Microinjection of RO60-0175 into the VTA blocks behavioral effects of cocaine (Fletcher,Chintoh, Sinyard, & Higgins, 2004), perhaps reflecting inhibition of DA cell firing and DArelease as noted earlier (Di Matteo et al., 2001). Microinjection of RO60-0175 into theprefrontal cortex also markedly reduces cocaine-induced locomotor activity (Filip &Cunningham, 2003), and this action may involve suppression of excitatory glutamate outputsto the nucleus accumbens (Liu, Bubar, Lanfranco, Hillman, & Cunningham, 2007).Neuroanatomical evidence suggests that effects of 5-HT2C receptor activation in the VTA andcortex are mediated by stimulation of γ-aminobutyric acid interneurons (Bubar & Cunningham,2006, 2007; Liu et al., 2007), and more research is needed to validate this proposal. Additionalinvestigation is warranted to fully elucidate the role of 5-HT2C receptors in mediatingantistimulant effects of 5-HT releasers. In addition, the potential of 5-HT2C agonists asmedications for substance use disorders deserves to be examined (Bubar & Cunningham,2006; Higgins & Fletcher, 2003).

Potential Adverse Effects of 5-HT ReleasersThe clinical use of 5-HT-releasing agents as medications has been associated with a numberof adverse effects (Rothman, Ayestas, Dersch, & Baumann, 1999; Rothman & Baumann,2002; Zolkowska, Rothman, & Baumann, 2006). Based primarily on experience with d,l-fenfluramine and its more potent isomer d-fenfluramine, three potentially serious side effectsneed to be considered when 5-HT releasers are developed as treatment agents: valvular heartdisease (VHD), idiopathic pulmonary arterial hypertension (IPAH), and neurotoxicity.Fenfluramines were commonly prescribed anorectics until their removal from the market in1997 because of the occurrence of VHD in some patients (Connolly et al., 1997; Connolly &McGoon, 1999). Fenfluramine-associated VHD is characterized by thickening of valve leafletsand increased regurgitation of blood, most often detected by echocardiography. While initialfindings suggested fenfluramines induce VHD in a high percentage of patients, more recentevidence shows a much smaller risk. For example, a meta-analysis of available clinical datademonstrates that the incidence of clinically significant valvular regurgitation was 12% infenfluramine-treated patients versus 6% in untreated controls (Sachdev, Miller, Ryan, & Jollis,2002).

Because fenfluramines are potent 5-HT releasers (Baumann et al., 2000; Rothman, Clark,Partilla, & Baumann, 2003) and 5-HT has established mitogenic effects (Nemecek, Coughlin,Handley, & Moskowitz, 1986; Seuwen, Magnaldo, & Pouyssegur, 1988), investigators initiallyspeculated that serotonergic mechanisms might contribute to VHD (Connolly et al., 1997;Connolly & McGoon, 1999). To this end, we carried out an investigation to determine whether



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stereoisomers of fenfluramine, or the N-dealkylated metabolite norfenfluramine, might activatemitogenic 5-HT receptors (Rothman, Baumann et al., 2000). A number of other test drugs wereincluded in these experiments as positive and negative controls. “Positive controls” were ergotalkaloids known to increase the risk of VHD, such as methysergide, its active metabolitemethylergonovine, and ergotamine (Bana, MacNeal, LeCompte, Shah, & Graham, 1974;Bredberg, Eyjolfsdottir, Paalzow, Tfelt-Hansen, & Tfelt-Hansen, 1986; Hendrikx, Van Dorpe,Flameng, & Daenen, 1996). “Negative controls” were drugs that interact with monoaminetransporters but do not cause VHD, and these drugs included phentermine, fluoxetine, and itsmetabolite norfluoxetine. We also tested the antidepressant trazodone and its active metabolite,m-chlorophenylpiperazine (mCPP), as negative controls (Ishida et al., 1995; Otani et al.,1997). mCPP has agonist activity at a wide range of 5-HT receptor subtypes (Hoyer et al.,1994, 2002) and is capable of releasing neuronal 5-HT via a transporter-mediated mechanismsimilar to fenfluramines (Baumann et al., 2001; Baumann, Rutter, & Auerbach, 1993).

Our working hypothesis was that fenfluramines, norfenfluramines, and positive control drugswould share the ability to activate a mitogenic 5-HT receptor subtype expressed in heart valves,while the negative control drugs would not. An initial receptorome screen led to a detailedevaluation of the binding of these drugs to the 5-HT2 family of receptors (Rothman, Baumannet al., 2000). Table 2 reports binding data, and Table 3 reports the functional effects of thesecompounds at cloned human 5-HT2A, 5-HT2B, and 5-HT2C receptors. Of note, fenfluramineshave low affinity for all 5-HT2 receptor subtypes. By contrast, we found that norfenfluraminesdisplay high affinity and efficacy at the 5-HT2B receptor subtype (KI = 10–50 nM), consistentwith the findings of others (Fitzgerald et al., 2000; Porter et al., 1999). Methysergide acts as apartial agonist at the 5-HT2B receptor, while the metabolite methylergonovine has even greateraffinity and efficacy. Ergotamine is a potent partial agonist at the 5-HT2B receptor. Among thenegative control drugs tested, only mCPP exhibits agonist activity at the 5-HT2B site. It isnoteworthy that trazodone binds to the 5-HT2B receptor with moderate affinity, but functionsas an antagonist. Thus, when trazodone is metabolized to mCPP in vivo (Ishida et al., 1995;Otani et al., 1997), the 5-HT2B actions of mCPP are probably blocked by antagonist actionsof the parent compound.

Our results with the various positive control drugs strongly implicate the 5-HT2B receptor asa major culprit in the development of drug-induced VHD. Of note, accumulating data supportthis hypothesis (Fitzgerald et al., 2000; Porter et al., 1999; Roth, 2007; Setola & Roth, 2005).5-HT2B receptors are abundantly expressed on aortic and mitral valves (Fitzgerald et al.,2000), and these receptors are known to stimulate mitogenisis (Hafizi, Taylor, Chester, Allen,& Yacoub, 2000; Lopez-Ilasaca, 1998). Additional evidence for the role of 5-HT2B receptorsin drug-induced VHD is based on the effects of ergot medications such as carbergoline andpergolide. Both of these medications increase the risk of VHD in human patients and are alsopotent 5-HT2B receptor agonists (for review, see Roth, 2007). Setola and coworkers (2003)showed that the illicit amphetamine analog 3,4-methylenedioxymethamphetamine (MDMA)and its N-demethylated metabolite, 3,4-methylenedioxyamphetamine, are 5-HT2B receptoragonists. These drugs stimulate prolonged mitogenic responses in human valvular interstitialcells via activation of 5-HT2B receptors (Setola et al., 2003). As predicted by this study, a recentclinical report found that heavy MDMA users display a significantly higher incidence ofvalvular regurgitation when compared with control subjects (Droogmans et al., 2007). Moreclinical investigations are needed to clearly establish the link between illicit MDMA use andthe risk for developing VHD.

Epidemiological evidence indicates that fenfluramines increase the risk of developingidiopathic pulmonary arterial hypertension (IPAH), a debilitating and incurable disease(Abenhaim et al., 1996; Fishman, 1999). IPAH is characterized by pulmonary arterialvasoconstriction and hyperplasia that leads to severe hypertension. The pathogenesis of IPAH



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is complex and difficult to study, especially given the rarity of the disorder. Nonetheless,numerous investigations from our laboratory and others implicate the involvement of 5-HTand SERT proteins in the pathogenesis of IPAH (Eddahibi et al., 2006; MacLean, Herve,Eddahibi, & Adnot, 2000; Rothman et al., 1999). We demonstrated that SERT substrate activityis a common feature of drugs associated with IPAH, but not all substrates increase the risk forthe disease (Rothman et al., 1999). It is well established that blood platelets express SERTproteins identical to those expressed on neurons, and that platelet SERT accumulates nearly99% of circulating 5-HT into platelet storage (Ni & Watts, 2006). One hypothesis—the so-called “5-HT hypothesis”—has been invoked as a potential mechanism underlyingfenfluramine-induced IPAH (Fishman, 1999; MacLean et al., 2000). This hypothesis postulatesthat fenfluramines increase the risk for IPAH by stimulating SERT-mediated release of 5-HTfrom platelets, thereby elevating plasma levels of 5-HT. Persistent elevations in plasma 5-HTwould then cause pulmonary dysfunction. It is noteworthy that an analogous 5-HT hypothesishas been invoked to explain VHD, but as discussed already, drug-induced activation of 5-HT2B receptors seems to be the predominant mechanism involved in this disease.

A key prediction of the 5-HT hypothesis is that fenfluramine increases plasma 5-HT toconcentrations sufficient to produce vasoconstriction and mitogenesis, which then leads toserious side effects. Despite the widespread acceptance of the 5-HT hypothesis as anexplanation for fenfluramine-associated IPAH, the effects of fenfluramine and related agentson plasma 5-HT have received little attention. Studies conducted in the 1990s do not supportthe 5-HT hypothesis because they show that acute fenfluramine does not increase plasma 5-HT in rats, and chronic fenfluramine treatment lowers blood 5-HT in humans (Martin &Artigas, 1992; Rothman, Redmon et al., 2000). Given the uncertainties regarding validity ofthe 5-HT hypothesis, we assessed the acute effects of fenfluramine and other amphetamineson plasma levels of 5-HT in rats (Zolkowska et al., 2006). Specifically, we developed a novelmicrodialysis method to measure plasma levels of 5-HT in whole blood samples obtained fromconscious catheterized rats. Using this method, baseline plasma 5-HT levels in rats are 0.22nM, or about 1 nM when corrected for dialysis probe recovery, which is similar to plasma 5-HT concentrations measured in human subjects (Herve et al., 1995). Important to note, systemicadministration of fenfluramine, MDMA and other amphetamines evokes transient dose-dependent increases in plasma 5-HT ranging from 4 to 20 nM. The ability of drugs to increaseplasma 5-HT is directly correlated with their ability to increase SERT-mediated 5-HT releasein neurons, suggesting the involvement of platelet SERT proteins. These data show thatfenfluramine and other 5-HT releasers are able to acutely increase plasma 5-HT, but theabsolute levels of 5-HT are well below the concentrations required to contract pulmonaryarteries or stimulate mitogenesis (Cortijo et al., 1997; Eddahibi et al., 1999).

From a medication development standpoint, a more relevant issue is whether chronicadministration of 5-HT releasers can persistently increases plasma 5-HT. To address thisquestion, we used microdialysis methods to examine the effects of 2-week minipump infusionsof fenfluramine (3 and 10 mg/kg/day) or the 5-HT uptake blocker fluoxetine (3 and 10 mg/kg/day) on plasma levels of 5-HT in rats (Zolkowska, Baumann, & Rothman, 2008). In this study,chronic administration of fenfluramine, but not fluoxetine, caused two- to fourfold increasesin baseline dialysate 5-HT in blood. Given baseline plasma 5-HT concentrations of about 1nM, fenfluramine-induced increases in plasma 5-HT are less than 5 nM. The data in Figure 5show that chronic exposure to fenfluramine or fluoxetine markedly reduces the ability of acutefenfluramine to evoke increases in plasma 5-HT. Thus, chronic exposure to fenfluramineminimizes the surges in plasma 5-HT caused by acute administration of the drug. Chronicminipump infusions of fenfluramine and fluoxetine in rats give rise to steady-state blood levelsof drugs and their bioactive metabolites that are similar to those measured in human patientstaking prescribed doses of these medications (Lundmark, Reis, & Bengtsson, 2001; Rothman,



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Redmon et al., 2000). Thus, our rat model system is relevant to human patients takingfenfluramine or fluoxetine.

It has been shown that 5-HT provokes contraction of human pulmonary arteries atconcentrations ranging from 100 nM to 10 μmol/L (Cortijo et al., 1997). The thresholdconcentration of 5-HT required to stimulate mitogenic responses in cultured human pulmonaryartery smooth muscle cells is about 10 nM (Eddahibi et al., 2001; Marcos et al., 2003), whilemuch higher 5-HT concentrations are needed to stimulate mitogenic responses in rat pulmonaryartery smooth muscle cells (Eddahibi et al., 1999; Pitt et al., 1994). Because we found thatchronic fenfluramine elevates baseline plasma 5-HT to less than 5 nM in rats, it appears thatfenfluramine-induced increases in plasma 5-HT are below the concentrations known to causepulmonary side effects. Fenfluramine-induced elevations of plasma 5-HT are also much lowerthan those required to produce VHD in rats exposed to exogenous 5-HT (580–974 nM)(Gustafsson et al., 2005). Viewed collectively, these findings demonstrate that the 5-HThypothesis cannot explain the mechanism of fenfluramine-associated IPAH or VHD. Ofcourse, it is possible that two- to fourfold increases in plasma 5-HT could be enough to stimulatemitogenic responses in susceptible individuals and increase the risk of developing IPAH.However, this scenario seems unlikely since treatment with lithium or MAO inhibitorsproduces two- to fourfold increases in plasma 5-HT without increasing the risk for IPAH(Artigas et al., 1989; Celada, Dolera, Alvarez, & Artigas, 1992).

At the present time, the mechanisms underlying fenfluramine-associated IPAH remainenigmatic. One significant problem is that most animal models of IPAH require the inductionof hypoxia, which is not a major factor in humans. Furthermore, the etiology of fenfluramine-associated IPAH may differ substantially from that of nondrug-related IPAH. Despite thesecaveats, recent findings have provided novel hypotheses to explain how fenfluramine mightcause IPAH. For example, Launay and colleagues (Launay et al., 2002) have provided evidencethat activation of 5-HT2B receptors in the lung is critical to the development of IPAH in ahypoxic mouse model. Since the stereoisomers of norfenfluramine are potent and selective 5-HT2B agonists (Rothman, Baumann, et al., 2000), a role for 5-HT2B receptors seems feasible.On the other hand, a number of medications that produce VHD and activate 5-HT2B receptorsdo not increase the risk for IPAH—these medications include methysergide, ergotamine,pergolide, and cabergoline.

Additional evidence from our laboratory disputes a role for 5-HT2B sites in fenfluramine-associated IPAH (Rothman & Baumann, 2006). Aminorex is a SERT substrate that caused anepidemic of IPAH in the 1960s (Fishman, 1999; Gurtner, 1985), and case reports implicate therelated designer drug 4-methylaminorex as a cause of the disease (Gaine, Rubin, Kmetzo,Palevsky, & Traill, 2000). If 5-HT2B receptors are involved in the pathogenesis of drug-associated IPAH, then one would suspect aminorex to target 5-HT2B sites. While aminorexdoes interact with cloned human 5-HT2B receptors, the half maximal effective concentration(EC50) of the drug for 5-HT2B receptor activation (870 nM) is 30-fold higher than its EC50 forNE release (26.4 nM). Moreover, the activity of aminorex at 5-HT2B sites is nearly 50-foldless than that of d-norfenfluramine. It seems plausible that metabolites of aminorex may actmore potently at 5-HT2B receptors, and this possibility deserves to be examined. However, theavailable data argue against an important role for 5-HT2B receptors in the pathogenesis ofanorectic-associated IPAH.

Recent studies by Eddahibi et al. (2006) focus attention on 5-HT produced locally in the lungas a critical player in pathogenesis of IPAH. These investigators reported that endothelial cellsin the pulmonary microvasculature synthesize 5-HT, which is then released as a growth factor.It has been proposed that dysregulation of 5-HT production in endothelial cells, along withoverexpression of SERT by the pulmonary artery smooth muscle cells, contributes to



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hyperplasia observed in IPAH. Immunohistochemical studies show that pulmonarymicrovascular endothelium do not express SERT (Eddahibi et al., 2006), indicating thatfenfluramine cannot release 5-HT from these cells. Based on the findings of Eddahibi et al.,administration of the 5-HT precursor L-5-hydroxytryptophan (5-HTP) would be predicted toincrease 5-HT synthesis in pulmonary endothelial cells because this compound bypasses therate-limiting enzyme tryptophan hydroxylase. 5-HTP is a commonly used dietary supplementwith a well-established history of safety (Das, Bagchi, Bagchi, & Preuss, 2004), and its use isnot known to increase the risk of IPAH. Although speculative, this observation suggests thatan increase in 5-HT synthesis in the pulmonary microvascular endothelial cells may benecessary, but is not sufficient, to increase the risk of IPAH.

Experiments in laboratory animals show that high-dose administration of fenfluramine or d-fenfluramine can cause long-term depletion of 5-HT and loss of SERT binding sites in the brain(McCann, Seiden, Rubin, & Ricaurte, 1997; Zaczek et al., 1990). The persistent nature offenfluramine-induced 5-HT deficits in the CNS has been interpreted as evidence forneurotoxicity, although this hypothesis and its clinical relevance are still a matter of debate(Rose, Hunt, Collins, Hindmarsh, & Jenner, 1996; Rothman, Jayanthi, et al., 2003). Themechanisms underlying fenfluramine-induced 5-HT depletions are not well understood, butacute 5-HT release has been implicated because 5-HT uptake blockers and synthesis inhibitorscan prevent long-term 5-HT depletions (Halladay, Kirschner, Hesse, Fisher, & Wagner,2001; Steranka & Sanders-Bush, 1979). An important observation is that not all SERT subtratesdeplete 5-HT (Baumann et al., 2001; Cozzi, Frescas, Marona-Lewicka, Huang, & Nichols,1998; Nichols, Brewster, Johnson, Oberlender, & Riggs, 1990). As noted previously, mCPPinteracts with SERT to release 5-HT from neurons, and mCPP is equipotent with d-fenfluramine in this regard (Baumann et al., 2001; Baumann, Mash, & Staley, 1995; Eriksson,Engberg, Bing, & Nissbrandt, 1999). The data in Figure 6 demonstrate that repeated high-doseadministration of mCPP fails to affect postmortem tissue levels of 5-HT in rat brain, whereasfenfluramine causes profound loss of 5-HT. These data indicate that SERT-mediated 5-HTrelease is separable from long-term 5-HT depletion.

Elucidating the mechanisms responsible for adverse effects of 5-HT releasers will haveimportant implications for the future development of SERT substrates as pharmacotherapies.The findings reviewed here provide clues for designing dual DA/5-HT releasers devoid offenfluramine-like adverse effects. In particular, any lead drug molecule must lack 5-HT2Bagonist activity to prevent the risk of VHD. In addition, candidate drugs should be chemicallydistinct from the phenylethylamine structure shared by amphetamine-like agents, becausenonamphetamine 5-HT releasers have a reduced capacity for causing neurotoxic effects, andpossibly IPAH.

PAL-287: A Nonamphetamine DA/5-HT ReleaserBased in part on the above rationale, we sought to identify and characterize a nonamphetaminetransporter substrate that would release DA and 5-HT, without affecting release of NE. Afteran extensive evaluation of over 350 compounds, we found it impossible to dissociate NE- andDA-releasing properties, perhaps because of the phylogenetic similarities between NET andDAT. The first lead compound from our search was PAL-287 (1-napthyl-2-aminopropane; seestructure in Figure 1), a novel nonamphetamine monoamine releaser (Rothman et al., 2005).The in vitro potency of PAL-287 at releasing radiolabeled transmitters from DAT, NET, andSERT is 12.6 ± 0.4 nM, 11.1 ± 0.9 nM, and 3.4 ± 0.2 nM, respectively (see Table 2). Figure 7shows that administration of PAL-287 to rats increases extracellular 5-HT and DA in a dose-dependent manner, with larger effects on 5-HT compared with DA. Functional studies withcloned human 5-HT2 receptors reveal that PAL-287 is a full agonist at 5-HT2B receptors(EC50 = 40 nM) and 5-HT2A receptors (EC50 = 466 nM). The drug is a potent partial agonist



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at 5-HT2C receptor sites (EC50 = 2.3 nM, EMAX = 20%), an effect that suggests possibleanorectic actions of PAL-287 (Nilsson, 2006;Vickers, Clifton, Dourish, & Tecott, 1999). 5-HT2C agonist activity may also contribute to the minimal reinforcing properties of PAL-287despite potent DA releasing actions of the drug (see Czoty et al., 2002;Higgins & Fletcher,2003). The weaker potency of PAL-287 at 5-HT2A and 5-HT2B receptors, compared with itsactivity at SERT, suggests the drug may not activate 5-HT2A and 5-HT2B receptors in vivo.Experiments should be conducted to test this hypothesis.

PAL-287 produces minimal locomotor activation despite substantial elevations in extracellularDA (see Figure 7). In particular, the amount of ambulation produced by 3 mg/kg PAL-287 isone third the amount produced by 1 mg/kg d-amphetamine, even though both drug treatmentscause equivalent DA release. These data suggest that 5-HT-releasing properties of PAL-287limit the stimulant effects of concurrent DA release. Repeated high-dose administration ofPAL-287 to rats (18 mg/kg ip, q 2 hr, 3 doses) fails to affect brain tissue 5-HT levels whenassessed two weeks after injections, unlike d-methamphetamine (6.0 mg/kg ip, q 2hr, 3 doses)and MDMA (7.5 mg/kg ip, q 2 hr, 3 doses) which cause significant 5-HT depletions. The datain Figure 8 show that PAL-287 does not support self-administration behavior, and chronicadministration of the drug decreases cocaine self-administration in rhesus monkeys. A dose of1.0 mg/kg/hr PAL-287 significantly reduces both cocaine- and food-maintained responding,but the suppression of cocaine self-administration is somewhat greater than the reduction infood-maintained responding.

Our results with PAL-287 confirm the hypothesis that a nonamphetamine substrate at DATand SERT will release DA and 5-HT from neurons in vivo, be minimally reinforcing, and alsosuppress ongoing cocaine self-administration. PAL-287 displays a number of desirablequalities for a candidate treatment medication, including minimal locomotor activation, lackof long-term 5-HT neurotoxicity, and weak reinforcing effects. Future studies will be necessaryto determine the potential of PAL-287 for increasing the risk for VHD and IPAH, especiallygiven the 5-HT2B agonist effects of the drug. The present data with PAL-287 support the useof monoamine releasers as agonist medications for the treatment of stimulant addictions. Adose of 1.0 mg/kg/hr PAL-287 virtually eliminated cocaine self-administration in rhesusmonkeys by the end of the 7-day treatment, although this effect was not entirely selective forcocaine versus food. We also note that the role of NE in the actions of PAL-287 is an importantissue awaiting additional study (Rothman et al., 2001).

ConclusionsOur findings with PAL-287 in monkeys are similar to the suppression of cocaine self-administration produced by d-amphetamine, although d-amphetamine displays greaterselectivity in reducing cocaine self-administration as opposed to food-maintained responding(Negus & Mello, 2003a). Grabowski et al. (Grabowski et al., 2001; Grabowski, Shearer et al.,2004) showed that a slow-release formulation of d-amphetamine is effective in maintainingcocaine addicts in treatment and reducing illicit cocaine use. We predict that agents likePAL-287, which have mixed DA/5-HT releasing activity, will possess the therapeutic effectsof amphetamine-type monoamine releasers, while minimizing the adverse effects associatedthe phenethylamine structure. Based on observations that dual DA/5-HT releasers suppressalcohol ingestion (Halladay et al., 1999; Halladay et al., 2006; Yu et al., 1997), it seems thatPAL-287 or similar agents should be tested as potential treatments for alcohol addiction. Inaddition, combined treatment with DA releasers and 5-HT releasers blocks alcohol withdrawalseizures (Yu et al., 1997). Although further work remains to refine PAL-287, in particular toreduce its potency at 5-HT2B receptors, we believe that PAL-287 represents the prototype fora new generation of drugs that enhance monoamine release by acting as substrates at multipletransporters.



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While compounds like PAL-287 move slowly from the preclinical arena toward clinicaldevelopment, it is possible to test the concept of administering dual DA/5-HT releasers inhumans by implementing clinically available compounds. For example, the utility of DA/5-HT releasers as treatments for addictive disorders can be tested by administration of the DAreleaser d-amphetamine along with the 5-HT precursor 5-HTP. It is noteworthy that 5-HTPmust be coadministered with the peripheral decarboxylase inhibitor carbidopa to selectivelyincrease extracellular 5-HT in CNS (see Halladay et al., 2006). Moreover, the utility of DA/5-HT releasers could also be tested using phentermine and 5-HTP/carbidopa, a drug combinationwith predicted efficacy as an appetite suppressant (Rothman, in press). In summary, we suggestthat drugs with a mode of action similar to that of PAL-287 will provide neurochemicalnormalization therapy for stimulant addictions, and might also be useful for treating depression,obsessive–compulsive disorder, attention deficit hyperactivity disorder and obesity.

AcknowledgementsThis research was supported in part by the Intramural Research Program of the National Institutes of Health, NIDAand NIDA R01 DA12970 to Bruce E. Blough.

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Figure 1.Chemical structures of stimulants discussed in this article.


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