Non-Amine Dopamine Transporter Probe[3H]Tropoxene Distributes

to Dopamine-Rich Regions of Monkey BrainRICHARD DE LA GARZA, II,1 PETER C. MELTZER,2 AND BERTHA K. MADRAS1*

1Harvard Medical School, New England Regional Primate Research Center, Division of Neurochemistry,Southborough, Massachusetts

2Organix Inc., Woburn, Massachusetts

KEY WORDS monoamines; reuptake; cocaine; autoradiography; primates

ABSTRACT Drug development in psychopharmacology has adhered to the unwrit-ten precept that compounds targeting monoamine transporters must contain an aminenitrogen in the molecular structure. A series of non-amine-bearing aryloxatropanes thatare potent inhibitors of the dopamine transporter (DAT) challenged this precept. In thepresent study, we investigated the brain distribution of a selective, high-affinity DATnon-amine, [3H]tropoxene (2-carbomethoxy-3,4dichloro-3-aryl-8-oxabicyclo[3.2.1] oc-tene), which binds to the DAT in monkey striatum. The autoradiographic distribution of[3H]tropoxene was conducted in tissue sections of rhesus (Macaca mulatta) monkeybrain. Highest accumulation of the radioligand was detected in the putamen and caudatenucleus, with significant levels also observed in the nucleus accumbens and substantianigra. Moderate to low levels of [3H]tropoxene binding were noted in the hypothalamus,amygdala, ventral tegmental area, and thalamus. The distribution of [3H]tropoxene wasrestricted to brain regions previously identified as expressing DAT, and the relativedensities of [3H]tropoxene binding sites in various brain regions corresponded to thoseobserved with other selective monoamine radioligands for the DAT. This is the firstreport to demonstrate that transporter-selective compounds that bear no amine nitrogenin their structure bind selectively to brain regions rich in the transporter. The resultssupport our conclusion that an amine nitrogen is not necessary for compounds to bind tomonoamine transporters and distribute in brain according to the known distribution oftransporters. The findings provide further incentives to investigate the pharmacologicalpotential of transport inhibitors lacking an amine nitrogen in the molecular structure.Synapse 34:20–27, 1999. r 1999 Wiley-Liss, Inc.

INTRODUCTION

Dopamine (DA) neurotransmission is initiated by thepresynaptic release of DA and is partly terminated byreuptake through a protein located on the presynapticnerve terminal, termed the DA transporter (DAT: Coyleand Snyder, 1969; Horn, 1978, 1990). The DAT is animportant regulator of DA levels in the synapse, andthe ensuing magnitude of DA receptor stimulation (forreview, see Kuhar, 1998). In brain, the DAT is localizedalmost exclusively on DA neurons (Ciliax et al., 1995;Vaughn et al., 1996), and is a target of the widely usedtherapeutic drugs methylphenidate (Ritalint), bupro-pion (Wellbutrint, and Zybant), as well as the addictivedrug cocaine (Bergman et al., 1989; Madras et al.,1989a; Reith et al., 1986; Ritz et al., 1987; Spealman etal., 1989).

In the last decade, considerable effort has beenexpended on developing novel ligands for the DAT as

therapeutic agents, as probes to investigate the mecha-nism(s) by which compounds block the DAT (Carroll etal., 1991, 1992, 1997; Kuhar et al., 1998; Madras et al.,1990; Meltzer et al., 1993), and as imaging agents forDA neurons (Fischman et al., 1998, Meltzer et al.,1997a,b, 1998). DAT inhibitors have characteristicallybeen designed with an amine nitrogen incorporated inthe molecular structure, a restriction based on anuntested premise that inhibitors require an aminenitrogen to bind to a counterion on the target protein.Our laboratory recently challenged this premise bydeveloping a series of non-nitrogen bearing compoundsin which the amine nitrogen was replaced by an oxygen

Contract grant sponsor: NIDA; Contract grant numbers: DA 05857, DA 06303,DA 11558, DA 00304; Contract grant sponsor: NIMH; Contract grant number:MH 14275; Contract grant sponsor: NCRR; Contract grant number; RR 00168.

*Correspondence to: B.K. Madras, Ph.D., Harvard Medical School, NERPRC,Division of Neurochemistry, One Pine Hill Drive, Southborough, MA 01772-9102.

SYNAPSE 34:20–27 (1999)

r 1999 WILEY-LISS, INC.

in the molecular structure (aryloxatropanes). Thesecompounds, termed non-amines, were shown to bepotent inhibitors of the DAT (Madras et al., 1996;Meltzer et al., 1997). To explore the pharmacologicalpotential of this novel class of drugs, it is necessary todetermine whether they retain the full spectrum ofbiological properties displayed by their amine-bearingcounterparts. One important parameter to consider iswhether a non-amine can selectively localize in brainregions that express its target, the DAT. To this end, weinvestigated the distribution of the non-amine [3H]tro-poxene (Fig. 1a), a high-affinity, selective DAT inhibitorin nonhuman primate brain. The distribution of [3H]tro-poxene was compared to that of several monoamineprobes (Fig. 1b) that bind DAT with high affinity.

MATERIALS AND METHODS

Adult, rhesus monkey (Macaca mulatta) brains (n 53, 2 females, 1 male) were obtained from the brain bankof the New England Regional Primate Research Center.Brains were initially sectioned into 1-cm slabs andstored at 285°C. Prior to thin sectioning, tissue slabswere placed into a Reichert-Jung cryostat to equilibrate

with an operating temperature of 210°C. Coronalsections of 20 µm were cut and thaw-mounted ontogelatin-coated (‘‘subbed’’) slides, air dried, and stored in

Fig. 1. Comparison of the molecular structure and affinity ofspecific compounds for the dopamine transporter versus serotonintransporter. a: [3H]tropoxene, a non-amine, is a highly selective ligandfor the dopamine transporter. b: Four ligands, containing nitrogen intheir molecular structure, which are commonly utilized to identify thedopamine transporter.

Fig. 2. Autoradiographic distribution of the non-amine [3H]tropox-ene in tissue sections of rhesus monkey brain at level of the striatum(approximately Å 18.5). a: Total binding was measured with 1 nM[3H]tropoxene. b: Nonspecific binding was measured with 1 nM[3H]tropoxene in the presence of 50 µM cocaine. At this coronal plane,high levels of [3H]tropoxene binding sites are prominent in the caudatenucleus (Cd) and putamen (Put), and to a lesser extent in the nucleusaccumbens (NAS).

TABLE I. Autoradiographic distribution of a non-amine,[3H]tropoxene, in tissue sections of rhesus monkey brain (N 5 3)

Brain areaSpecific bindingpmol/g tis. eq.

Region:cerebellum ratio

Caudate 30.9 6 3.05 51:1Putamen 30.7 6 3.59 51:1Substantia nigra 15.2 6 1.56 25:1Nucleus accumbens 12.1 6 2.49 20:1Hypothalamus, median emi-

nence 9.94 6 2.71 17:1Ventral tegmental area 3.64 6 1.25 6:1Hypothalamus, ventral medial

nucleus 3.25 6 0.85 5:1Hypothalamus, dorsal medial

nucleus 2.95 6 0.91 5:1Stria terminalis 2.58 6 1.03 4:1Globus pallidus 2.53 6 0.75 4:1Thalamus, ventral anterior

nucleus 2.38 6 0.89 4:1Hippocampus 1.91 6 0.85 3:1Amygdala 1.22 6 0.51 2:1Cerebellum 0.60 6 0.42 1Medial Pre Frontal Cortex 0.57 6 0.30 1Pons 0.19 6 0.13 0.3:1Septum 0.14 6 0.14 0.2:1Internal Capsule 0.09 6 0.08 0.1:1White Matter 0 0

Distribution of [3H]tropoxene was measured as described in Materials andMethods. Results are expressed as pmol/g tissue equivalents (mean 6 S.E.M.).Brain areas were measured in triplicate for each individual.

21BRAIN DISTRIBUTION OF NONAMINE [3H]TROPOXENE

sealed containers with desiccant capsules at 220°Cuntil use. Coronal tissue sections (20 µm) containingthe striatum (caudate nucleus and putamen) were usedin parametric studies to establish the appropriateconditions for autoradiography. Total binding was deter-mined using 1 nM [3H]tropoxene (spec. act. 87 Ci/mmol)and nonspecific binding was determined with 50 µMcocaine. Tissue sections (three replicates from eightanterior–posterior planes for each brain) were preincu-bated with Tris NaCl buffer (50 mM Tris HCl; 120 mMNaCl, pH 7.4 at 4°C) for 30 min, followed by incubationin Tris NaCl buffer containing 1 nM [3H]tropoxenealone or in the presence of 50 µM cocaine for 18 h at 4°C.Afterward, sections were washed twice (30 sec each) inTris HCl buffer (50 mM Tris HCl, pH 7.4, at 4°C),followed by a 2-sec dip in ice-cold double-distilled water.Sections were dried rapidly with a stream of chilled,

desiccated air. Slide-mounted tissue sections, alongwith calibrated standards ([3H]Microscales; Amer-sham, Arlington Heights, IL), were placed in X-raycassettes. [3H]Hyperfilm (Amersham) was apposed tothe slides and the cassettes were sealed, then stored at4°C in opaque containers with desiccant capsules for 35days.

The distribution and levels of radioactivity in brainwere determined with the MCID Image Analysis Sys-tem (Imaging Research, St. Catharines, Ontario). At-lases of rhesus monkey brain (Snider and Lee, 1961;Krieg, 1975) were used to establish neuroanatomicallandmarks on the autoradiograms. Relative opticaldensities of the standard-exposed films were fitted bylinear interpolation in order to convert optical densitiesof the tritium-exposed films to tissue equivalent bind-ing densities of [3H]tropoxene.

Fig. 3. [3H]Tropoxene accumulation in three planes of the caudatenucleus (a,c,e) and putamen (b,d,f). Medial-to-lateral and dorsal-to-ventral analyses were performed at approximately Å 18.5, where eachmeasurement (a–d: 1–8) represents a rectangular target approxi-mately 6 mm in diameter. Anterior-to-posterior analyses were per-formed along four successive coronal planes, as shown (e,f). In thecaudate, a progressive decrease in [3H]tropoxene binding sites was

observed along the medial-to-lateral plane (a), whereas an increasewas noted in the putamen (b). Along the dorsal-to-ventral plane, aninverted-U change in density was observed in the caudate (c), but nochange was observed in the putamen (d). Anterior-to-posterior gradi-ents were observed in the caudate (e), and were more robust in theputamen (f).

22 DE LA GARZA ET AL.

Custom synthesis of [3H]tropoxene was kindly pro-vided by Dr. S. Hurt of Dupont-New England Nuclearby insertion of tritium as shown in Table I. TheNational Institute on Drug Abuse provided (-)-cocainehydrochloride.

RESULTS

Initially, parametric studies were performed in tissuesections of brain to establish optimal conditions for[3H]tropoxene in vitro autoradiography. An association(incubation) time of 18 h and a dissociation (bufferrinse) time of 60 sec were chosen for use in this studysince shorter incubation times and longer buffer rinsetimes resulted in incrementally lower total and nonspe-cific values below equilibrium (data not shown). Theconcentration of [3H]tropoxene, 1 nM, was chosen be-cause it is close to the apparent KD value for [3H]tropox-ene measured in tissue homogenates (Madras et al., inpreparation). Autoradiography was conducted in tripli-cate with adjacent coronal sections (20 µm) to measuretotal and nonspecific binding. Specific binding of 55–60% was obtained with 1 nM [3H]tropoxene whenmeasured in the presence of the competing drug 50 µMcocaine. Analysis of the autoradiograms revealed high-est densities (.30 pmol/g tissue equivalents) of [3H]tro-poxene binding sites in the caudate nucleus and puta-men (Table I; Fig. 2). The distribution of binding siteswas not homogeneous in the caudate nucleus andputamen. In these brain regions, gradients in bindingsite densities were observed in medial-to-lateral, dorsal-to-ventral, and anterior-to-posterior planes. Specifi-cally, in the caudate nucleus a progressive decrease in[3H]tropoxene binding sites was observed along themedial-to-lateral plane (Fig. 3a), whereas an increasewas noted in the putamen (Fig. 3b). Analysis along thedorsal-to-ventral plane revealed a change in density(inverted-U function) in the caudate, but no change inthe putamen (Fig. 3c,d, respectively). Anterior-to-posterior gradients were observed in the caudate, andwere more robust in the putamen (Fig. 3e,f, respec-tively).

Although the caudate and putamen contained thehighest levels of [3H]tropoxene, other regions also accu-mulated significant levels. The nucleus accumbens(Fig. 4b; Table I) contained approximately 39% of theoverall binding sites (tissue equivalents) found in thecaudate nucleus, while the substantia nigra (Fig. 4f)contained approximately 49% of the overall bindingsites found in the caudate. The discrete localization of[3H]tropoxene to the DA-rich caudate, putamen, nucleusaccumbens, and substantia nigra, suggests that thisligand selectively targets DAT in the brain.

Lower levels (2–10 pmol/g) of [3H]tropoxene bindingsites were detected in the median eminence, dorsal andventral medial nuclei of the hypothalamus, stria termi-nalis (Fig. 4d), ventral anterior nucleus of the thalamus(Fig. 4e), globus pallidus (Fig. 4c), and ventral tegmen-

tal area (Fig. 4f). Low levels (,2 pmol/g) of [3H]tropox-ene binding sites were found in the hippocampus (Fig.4f) and amygdala (Fig. 4c). Binding sites labeled by[3H]tropoxene were near background levels (,0.60pmol/g) in all other regions identified (Table I), includ-ing the cerebellum (Fig. 4h), medial-prefrontal cortex(Fig. 4a), septum (Fig. 4b), and pons (Fig. 4g). Thesesites displayed 0.2–1.8% of the total binding for [3H]tro-poxene observed in the caudate nucleus.

DISCUSSION

Non-amines are a novel class of transporter drugsthat, unlike conventional transporter or receptor agents,bear no amine nitrogen in their structure. The presentresults demonstrate that a non-amine [3H]tropoxenedistributes in brain according to the known distributionof the DAT previously determined with monoaminetransporter probes. The results support our conclusionthat an amine nitrogen is not necessary for compoundsto selectively bind to and to distribute in these brainsites.

The distribution of [3H]tropoxene in brain corre-sponded closely to the distribution observed with selec-tive DAT probes that contain an amine nitrogen in theirstructure, including [3H]CFT (Canfield et al., 1990;Kaufman et al., 1991; Madras et al., 1989a,b), [125I]altro-pane (Madras et al., 1998a,b), [125I]RTI-121 (Little etal., 1995; Staley et al., 1995), and [125I]RTI-55 (Cline etal., 1992; Staley et al., 1994, 1995). The distribution of[3H]tropoxene also corresponded with the brain distri-bution of the DAT previously detected with antibodies(Ciliax et al., 1995; Freed et al., 1995; Nirenberg et al.,1996, 1997a,b) to the DAT.

Figure 5 presents the comparison of the brain distri-bution of [3H]tropoxene with four monoamine radioli-gands, which include DAT (vs. serotonin transporter:SERT) -selective tropanes ([3H]CFT, [125I]altropane,and [125I]RTI-121), and the nonselective tropane[125I]RTI-55 (see Fig. 1b). Among the radioligands pre-sented in the comparison in Figure 5, [125I]RTI-55 is theleast selective (DA:SERT) and displays high affinity forsites associated with the SERT (Staley et al., 1994; Bojaet al., 1991, 1992; Kaufman and Madras, 1992; Little etal., 1993; Cline et al., 1992; Coulter et al., 1995). All fiveradioligands revealed highest densities of binding sitesin the caudate nucleus and putamen, and lower levelsin the nucleus accumbens and substantia nigra. Both[125I]RTI-121 and [125I]RTI-55 revealed higher levels ofbinding in the nucleus accumbens than detected withthe more selective probes [3H]tropoxene, [3H]CFT, or[125I]altropane. For substantia nigra, [125I]RTI-55 and[3H]tropoxene revealed higher levels of binding thandemonstrated with [3H]CFT, [125I]altropane, or [125I]RTI-121. Relative densities of binding sites in all other brainregions included in the comparison (i.e., globus palli-dus, hippocampus, thalamus, and cerebellum) weregenerally similar (low to nondetectable) for all five

23BRAIN DISTRIBUTION OF NONAMINE [3H]TROPOXENE

Fig. 4

24 DE LA GARZA ET AL.

radioligands. Thus, it can be concluded that an impor-tant property for selective monoamine probes for accu-mulation in selective brain regions is shared by anon-amine.

In the hypothalamus, [3H]tropoxene binding siteswere detected in specific subnuclei of the hypothala-mus, including the dorsal and ventral medial nucleus,and median eminence (Table I). It is not possible tocompare these findings with monoamine probe densi-ties in the hypothalamic subnuclei, since values re-ported in other studies likely accounted for measure-ments of the entire region of hypothalamus. Theimportance of distinguishing between general hypo-thalamus and specific subnuclei of the hypothalamushas previously been shown (Meister and Elde, 1993;Demarest and Moore, 1979; George and Van Loon,1982).

[3H]Tropoxene binding in the hypothalamus may bespecifically associated with the DAT. While present in

Fig. 4. Autoradiographic distribution of total [3H]tropoxene bind-ing sites in rhesus monkey brain from eight anterior to posteriorplanes (n 5 3). Coronal tissue sections were incubated for 18 h at0–4°C, washed in buffer, and opposed to tritium-sensitive film, asdescribed in Materials and Methods. The autoradiograms were devel-oped 35 days later. A representative example is given in this figure. a:At the most anterior level, approximately Å 35.0, no significantlabeling was observed in the prefrontal or medial prefrontal cortex(mPFC). b: At approximately Å 18.5, intense labeling (dark gray toblack) of the caudate nucleus (Cd) and putamen (Put) is observed.High levels of [3H]tropoxene binding sites are observed in the nucleusaccumbens (NAS), but not in septum (Sep). c: At approximately Å 17.0,intense labeling of the caudate nucleus and putamen is observed, withlittle labeling of the globus pallidus (GP) or amygdala (Amy). d: Atapproximately Å 11.0, intense labeling of the caudate nucleus andputamen is still prevalent, and some labeling in amygdala andsubnuclei of the hypothalamus (Hypo). e,f: At approximately Å 10.0and 8.5, respectively, intense labeling of the caudate nucleus (headand tail) and putamen are prominent, and moderate levels areapparent in the substantia nigra (SN). Low levels were observed in thehippocampus (Hip), thalamus (Thal), and ventral tegmental area(VTA). g: At approximately Å 1.5, a fragment of the tail of the caudatenucleus is still apparent, with low levels in surrounding brainstructures, including the pons. h: At the most posterior plane, atapproximately P 4.0, the cerebellum (Cbl) and surrounding cortex areat very low levels.

Fig. 5. Comparison of primate brain distribution of a non-amineprobe for the dopamine transporter, [3H]tropoxene, with other mono-amine probes. The five radioligands revealed highest binding sitedensities in the caudate nucleus and putamen. Significant, but lower,levels are observed in the nucleus accumbens and substantia nigra.Relative densities of binding sites in all other brain regions included inthe comparison were generally similar (low to nondetectable) for all

five radioligands. All data were derived from in vitro autoradiogramsof primate brain tissues. Densities in various brain regions werenormalized by expressing values as percent specific binding of thecaudate nucleus (5 100%). Data for [3H]tropoxene is from the currentstudy, for [3H]CFT from Kaufman et al. (1991), for [125I]altropane fromMadras et al. (1998b), for [125I]RTI-121 from Staley et al. (1995), andfor [125I] RTI-55 from Staley et al. (1994).

25BRAIN DISTRIBUTION OF NONAMINE [3H]TROPOXENE

considerably lower numbers than ventral midbrainnuclei, DAT mRNA has been localized to several hypo-thalamic nuclei (Cerruti et al., 1993; Maggos et al.,1997; Meister and Elde, 1993; Revay et al., 1996;Shimada et al., 1994), and uptake of [3H]DA has beenshown in subnuclei of the hypothalamus (Demarest andMoore, 1979; George and Van Loon, 1982). Importantly,the presence of DAT mRNA, and [3H]DA uptake, corre-sponds with the hypothalamic subnuclei identified inthe current study. A thorough understanding of the roleof the DAT in hypothalamic nuclei is not known, butone function may be in regulating developmental stagesin the pituitary gland, since DAT-deficient mice developanterior pituitary hypoplasia and dwarfism (Bosse etal., 1997).

Moderate levels of DAT binding in the hypothalamushave been previously reported using the nonselectiveradioligand [125I]RTI-55 (Staley et al., 1994). As thesewere significantly reduced by pretreatment with theselective serotonin reuptake inhibitor citalopram, thosesites were predicted to be associated with the SERT. Itis possible that the hypothalamic nuclei identified with[3H]tropoxene are also of serotonergic origin, but defi-nite conclusions regarding the identity of the sitescannot be drawn in the present study.

The high specificity of [3H]tropoxene for the DATsuggests that the absence of an amine nitrogen incompounds targeted to transporters need not affect thespecificity of the compound or its distribution in brain.The evidence of non-amines as pharmacologically ac-tive agents is mounting. Non-amines have an in vivoprofile of effects that are similar to DAT inhibitors. Inthis regard, they increase extracellular DAlevels, engen-der cocaine-like discriminative stimulus effects (Ma-dras et al., in preparation), and distribute to DA-richbrain regions (current study). The predominance of[3H]tropoxene binding sites in the striatum is consis-tent with its high affinity for the DAT. The DAT in thesebrain regions, and in particular the nucleus accumbens,is thought to contribute to the locomotor (Kalivas andDuffy, 1993a,b), discriminative stimulus (Callahan etal., 1994), and reinforcing (Moore et al., 1998; Caine etal., 1995; Maldonado et al., 1993; Pettit and Justice,1989) effects produced by cocaine.

The most important conclusion from this and previ-ous studies (Madras et al., 1996) is that the presence ofan amine nitrogen in a compound is not a prerequisitefor compounds to possess pharmacological effects on theDAT or to accumulate selectively in brain regionsassociated with the DAT. The results have significantimplications for drug discovery, as all monoamine trans-porters previously designed to target transporters con-tain an amine nitrogen in the molecular structure. Ithas long been assumed that the presence of an amine isan indispensable component to confer affinity to itstarget, in analogy with endogenous amines (e.g., DA,SERT). The mechanism proposed was an ionic interac-

tion of an aspartic acid residue (Asp79) of the DATprotein with the amine nitrogen of the target compoundor dopamine (Kitayama et al., 1993). As previouslyshown in this laboratory, formation of an ionic bond isnot possible with this 8-oxa compound (or 8-carbanon-amines) and yet it binds to the DAT and blockstransport with a potency similar to its nitrogen-bearingcounterparts (Madras et al., 1996). An added advantageof this particular structure, [3H]tropoxene, is its highselectivity for the DAT over the SERT transporter (Fig.1a,b). The proposed advantage of developing com-pounds without a nitrogen in the molecular structure isthat these novel compounds might possess uniquebiological profiles that confer advantages over theirnitrogen-bearing counterparts. These may include adistinct mode of binding to the DAT, and differences intheir kinetic profile, metabolism, and elimination path-ways (Madras et al., 1996; Meltzer et al., 1997).

In summary, the non-amine [3H]tropoxene, a potentinhibitor of the DAT, selectively targets brain regionsrich in the DAT viewed by autoradiography. The distri-bution pattern of this novel non-amine probe is similarto that previously described with other monoamineprobes for the DAT. The striking and unequivocalresults reported here corroborate and support our viewthat the necessity of an amine nitrogen for compoundsto bind the DAT is unfounded. It is apparent thatexamination of compounds lacking an amine nitrogenin their molecular structure is a viable avenue forfuture drug development.

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27BRAIN DISTRIBUTION OF NONAMINE [3H]TROPOXENE