Peter C. Meltzer

Rapid detection of Parkinson's disease by SPECT with altropane: A selective ligand for dopamine transporters

Increasing evidence indicates that dopamine (DA) transporter density declines in Parkinsons disease (PD). 2β‐Carbomethoxy‐3β‐(4‐fluorophenyl)‐n‐(1‐iodoprop‐1‐en‐3‐yl) nortropane (IACFT, Altropane™) is a cocaine analog with high affinity and selectivity for dopamine transporter (DAT) sites in the striatum. In this study, single photon emission computed tomography (SPECT) with [123I]altropane was used to measure DAT density in seven healthy volunteers (five males, age 37–75, and two females, ages 26 and 39) and eight male patients with Parkinsons disease (age 14–79, Hoehn and Yahr stage: 1.5–3 (n = 5) and 4–5 (n = 3)). Dynamic SPECT images and arterial blood samples were acquired over 1.5–2 hr and plasma radioactivity was analyzed chromatographically to obtain metabolite corrected arterial input functions. Binding potential (BP, B′max/KD) for striatal (Str) DAT sites was calculated by two methods using occipital cortex (Occ) as a reference. In the first method, tissue time–activity curves (TAC) and metabolite corrected arterial input functions were analyzed by a linear graphical method developed for reversible receptor ligands. In the second method, the expression (StrTAC − OccTAC) was fitted to a gamma variate function and the maximum divided by OccTAC at the same time was used to estimate BP. In five of the PD patients, the SPECT data were compared with the results of PET with [18F] 6‐fluoro DOPA (FD‐PET). Plasma analysis indicated that [123I]altropane is rapidly converted to polar metabolites. SPECT images in healthy volunteers showed that [123I]altropane accumulated rapidly and selectively in the striatum and yielded excellent quality images within 1 h after injection. Both methods of analysis revealed a 7.6%/decade reduction in BP and average striatal values (corrected to age 25) were 1.83 ± 0.22 and 2.09 ± 0.20 by methods 1 and 2. In all the PD patients, striatal accumulation was markedly reduced and the pattern of loss was similar to that reported for DA; most profound in the posterior putamen with relative sparing of the caudate nuclei. A comparable pattern was observed with FD‐PET. For total striatum, age‐corrected BP was significantly (P < 0.001) reduced; 0.83 ± 0.06 (method 1), 0.84 ± 0.07 (method 2). BPs measured by the two methods were remarkably similar and highly correlated r2 = 0.88, (P < 0.001). These results indicate that [123I]altropane is an excellent SPECT ligand for imaging the DAT/DA neurons in human brain. The high selectivity and rapid striatal accumulation of the ligand allows for accurate quantitation of DAT sites in less than 2 hr. The results further demonstrate that [123I]altropane is an effective marker for PD. Synapse 29:128–141, 1998.

Further Evidence of Dopamine Transporter Dysregulation in ADHD: A Controlled PET Imaging Study Using Altropane

BACKGROUND The dopamine transporter (DAT) is known to be a key regulator of dopamine, and recent studies of genetics, treatment, and imaging have highlighted the role of DAT in attention-deficit/hyperactivity disorder (ADHD). The findings of in vivo neuroimaging of DAT in ADHD have been somewhat discrepant, however. METHOD Dopamine transporter binding was measured using a highly selective ligand (C-11 altropane) and positron emission tomography (PET). The sample consisted of 47 well-characterized, treatment-naïve, nonsmoking, non-comorbid adults with and without ADHD. Additionally, control subjects had few symptoms of ADHD. RESULTS Results showed significantly increased DAT binding in the right caudate in adults with ADHD compared with matched control subjects without this disorder. CONCLUSIONS These results confirm abnormal DAT binding in the striatum of adults with ADHD and provide further support that dysregulation of DAT may be an important component of the pathophysiology of ADHD.

Technepine: A high-affinity 99mtechnetium probe to label the dopamine transporter in brain by SPECT imaging

Increasing evidence suggests that the dopamine transporter, localized on dopamine neurons, is a marker for a number of physiological and pathological states (KaufmanandMadras, 1991,1993; Madras et al., 1990a, Schoemaker et al., 1985; Singer et al., 1991). With the development of sensitive probes, brainimaging and mea- surement of the transporter have become feasible in re- cent years (Brownell et al., in press; Innis et al., 1991; Frost et al., 1993; Madras et al., 1991; Morns et al., sub- mitted; Seibyl et al., 1995; van Dyke et a1.,1995; Wonget al., 1993,1995). Drugs of many chemical classes, includ- ing cocaine, bind to the dopamine transporter (Seeman, 1993). Nevertheless, effective imaging agents have been developed almost exclusively from the phenyltropane analogue of cocaine WIN 35,428 or CFT, a potent dopa- mine transport inhibitor (Clarke et al., 1973; Heikkila et al., 1979). The impetus for developing [llC]WIN 35,428 as a PET ligand (Hantraye et al., 1992; Madras, 1994; Madras et al., 1991, 1994; Wong et al., 1993; Meltzer et al., 1993) and y-emitting analogues for SPECT imaging (e.g., RTI-55, the 4-iodophenyl analogue of WIN 35,428, Canfield et al., 1990; Boja et al., 1991; Innis et al., 1991) arose directly from our observations of the binding of WIN 35,428 to the dopamine transporter. Unlike previ- ous dopamine transport inhibitors (noncocaine conge- ners) proposed for brain imaging(Kuhar et a1.,1990), the radiolabeled form of WIN 35,428 binds to the dopamine transporter in brain striatum with very low levels of non- specific binding (Madras et al., 1989a,b) and distributes principally to dopamine-rich regions of brain, as we re- ported in 1989 (Canfield et al., 1989) and subsequently (Canfield et al., 1990; Kaufman et al., 1991; Kaufman and Madras, 1992). SPECT imaging techniques are more practical than PET for routine clinical studies because of the lesser

Altropane, a SPECT or PET imaging probe for dopamine neurons: III. Human dopamine transporter in postmortem normal and Parkinson's diseased brain

Increasing evidence suggests that the dopamine transporter is situated almost exclusively on dopamine neurons. Accordingly, it is an valuable marker for Parkinsons disease and other pathological states of dopamine neurons. We previously demonstrated that the potent dopamine transport inhibitor [125I]altropane (IACFT:E‐N‐iodoallyl‐2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane) is a high affinity selective probe for the dopamine transporter in monkey brain and an effective SPECT imaging agent in nonhuman primate brain. We now report the binding properties of [125I]altropane in postmortem tissue of normal human brain and compare the findings to Parkinsons diseased brain. In homogenates of human brain putamen, [125I]altropane bound with high affinity (KD: 4.96 ± 0.38 nM, n = 4) and site density (BMAX: 212 ± 41.1 pmol/g original wet tissue weight) well within the density range reported previously for the dopamine transporter in this brain region. Drugs inhibited [125I]altropane binding with a rank order of potency that corresponded closely to their rank order for blocking dopamine transport (r 0.98, P < 0.001). In postmortem Parkinsons diseased brain, bound [125I]altropane (1 nM) was markedly reduced (89%, 99% in putamen, depending on measures of nonspecific binding) compared with normal aged‐matched controls (normal putamen: 49.2 ± 8.1 pmol/g; Parkinsons diseased putamen: 0.48 ± 0.33 pmol/g; n = 4). In vitro autoradiography, conducted in tissue sections at a single plane of the basal ganglia, revealed high levels of [125I]altropane binding the caudate nucleus and putamen, but lower levels (73% of the caudate‐putamen) in the nucleus accumbens (n = 7). In Parkinsons diseased brains (n = 4), [125I]altropane binding was 13% of the levels detected in normal putamen, 17% of normal values in the caudate nucleus, and 25% of normal levels in nucleus accumbens. The association of [125I]altropane to the dopamine transporter in human postmortem tissue, the marked reduction of [125I]altropane binding in Parkinsons diseased brains, its rapid entry into brain and highly localized distribution in dopamine‐rich brain regions, support its use as a probe for monitoring the dopamine transporter in vitro and in vivo by SPECT imaging. Synapse 29:116–127, 1998.

[11C,127I] Altropane: A highly selective ligand for PET imaging of dopamine transporter sites

The E isomer of 123I‐2β‐carbomethoxy‐3β‐(4‐fluorophenyl)‐N‐(1‐iodoprop‐1‐en‐3‐yl)nortropane (Altropane®) shows high affinity (IC50 = 6.62 ± 0.78 nmol) and selectivity (DA/5‐HT = 25) for DAT sites in the striatum. Recently, dynamic SPECT studies in healthy volunteers and patients with Parkinson disease demonstrated that the kinetics of striatal accumulation followed a pattern that is characteristic of a reversible tracer with maximal accumulation within 30 min after injection. These findings suggested that radiolabeling Altropane with [11C] might provide an equivalent and complementary tracer for PET studies. [127I] Altropane was treated with HCl to hydrolyze the methyl ester bond and yield a precursor for [11C] labeling. Introduction of an [11C] methyl ester group was achieved by treatment with [11C] CH3I followed by HPLC purification. Five healthy rhesus monkeys were injected with ∼10 mCi of [127I,11C] Altropane and dynamic PET images were acquired over 90 min. Arterial blood samples were collected in parallel with imaging and metabolite analysis was performed by HPLC. The PET and metabolite corrected arterial blood data were to calculate k3/k4 by two methods: 1) nonlinear least‐squares fitting, and 2) a linear graphical method for reversible ligands. The synthetic procedure yielded high specific activity tracer, >1,000 mCi/μ mole, with radiochemical purity >95%. Synthesis time was ∼30 min. The PET images revealed excellent striatal definition, with clear separation of caudate nucleus and putamen and minimal accumulation in brain regions with high 5HT transporter density. Metabolite analysis demonstrated that at 60 min after injection, ∼80% of circulating tracer was intact [127I,11C] Altropane and the remainder was converted to polar metabolites. Values for k3/k4 calculated by two analysis methods were remarkably similar: Method 1, 3.48 ± 0.41; Method 2, 3.77 ± 0.45 (mean ± SEM, t = 2.31, df = 8, P = 0.64). These results establish that Altropane has the important characteristics of: 1) rapid and specific striatal binding; 2) high selectivity for DA vs. 5‐HT transporter sites; 3) reversible binding kinetics; 4) potential for multiple injection studies; 5) high efficiency labeling with either [11C] or [123I]; 6) applicability for both PET and SPECT. These properties make Altropane an important DAT ligand for both research and clinical applications. Synapse 39:332–342, 2001.

Nitrogen‐based drugs are not essential for blockade of monoamine transporters

In brain, monoamine transporters are principal targets of widely used therapeutic drugs including antidepressants, methylphenidate (Ritalin), and the addictive drug cocaine. Without exception, these transport blocking agents contain an amine nitrogen. A prevalent view and untested promise is that an amino nitrogen is needed to bind to the same counterion on the transporter as does the amino nitrogen of the monoamine neurotransmitter. We report that several compounds without nitrogen (8‐oxa‐bicyclo‐3‐aryl‐[3.2.1] octanes, or aryloxatropanes) are active at monoamine transporters. One of these, tropoxane (O‐914), bound with high affinity to the dopamine (IC???: 3.35 ± 0.39 nM), serotonin (IC??? 6.52 ± 2.05 nM), and norepinophrine (IC???: 20.0 ± 0.3 nM) transporters in monkey brain, the human striatal dopamine transporter (IC???: 5.01 ± 1.74 nM), and blocked dopamine transport (IC???: 7.2 ± 3.0 nM) in COS‐7 cells transfected with the human dopamine transporter. These unique compounds require a revision of current concepts of the drug binding domains on monoamine transporters, open avenues for discovery of a new generation of drugs and raise the issue of whether mammalian transporters and receptors may respond to, as yet, undiscovered non‐amine bearing neurotransmitters or drugs.

Altropane, a SPECT or PET imaging probe for dopamine neurons: II. distribution to dopamine-rich regions of primate brain

The dopamine transporter in brain, localized almost exclusively on dopamine neurons, is an effective window on dopamine neurons. SPECT or PET imaging of the transporter in brain requires selective imaging agents that display appropriate pharmacokinetic properties. We previously reported that [125I]altropane ([125I]IACFT,2β‐carbomethoxy‐3β‐(4‐fluorophenyl)‐n‐ (1‐iodoprop‐1‐en‐3‐yl)nortropane) bound with high affinity (Kd: 5.33 nM) to a single site on the dopamine transporter and was selective for dopamine over the serotonin transporter in homogenates of monkey striatum. To determine whether the selective binding of [125I]altropane is reflected in its brain distribution, the in vitro and ex vivo distribution of [125I]altropane in squirrel monkey (Saimiri sciureus) brain was determined by quantitative autoradiography of coronal brain sections. In vitro, [125I]altropane (2 nM) distribution was discrete and was detectable primarily in the dopamine‐rich putamen, caudate nucleus, and nucleus accumbens. The resulting putamen:cerebellum ratio exceeded 120:1 (n = 3). The selective in vitro binding of [125I]altropane to the dopamine transporter, at concentrations approaching its Kd value (Kd: 5.33 nM, a single high affinity site), highlight its suitability for investigating the density of the dopamine transporter in various brain regions in vitro. Ex vivo autoradiography was conducted in monkeys to determine whether the brain distribution of [125I]altropane in vitro was predictive of its brain distribution pattern after intravenous administration. Thirty minutes after intravenous injection, highest levels of [125I]altropane (0.3 nmol/kg) were detected in the caudate‐putamen and nucleus accumbens and lowest levels in the cerebellum and cortex. The putamen or caudate:cerebellum ratio was 7. SPECT imaging of the brain within 30 min of i.v. injection confirmed the rapid and selective accumulation of [123I]altropane to the striatum. The selective binding of altropane to the dopamine‐rich striatum within 30 min of i.v. administration indicates that it is uniquely suited for SPECT or PET imaging of the dopamine transporter and associated dopamine neurons. Synapse 29:105–115, 1998.

Altropane, a SPECT or PET imaging probe for dopamine neurons: I. dopamine transporter binding in primate brain

Increasing evidence suggests that the dopamine transporter is an important marker for physiological and pathological changes in dopamine neurons. Potent dopamine transport inhibitors of the phenyltropane series (e.g., WIN 35,428 or CFT) are particularly suitable for PET (positron emission tomography) or SPECT (single photon emission computed tomography) imaging of the dopamine transporter in living brain. We investigated whether altropane, an N‐iodoallyl analog of WIN 35,428 (IACFT:E‐N‐iodoallyl‐2β‐carbomethoxy‐3β‐(4‐fluorophenyl)tropane), displayed in vitro properties suitable for evaluation as a SPECT imaging agent. In brain striatum of cynomolgus monkey (Macaca fascicularis), the unlabeled E‐isomer (IC50: 6.62 ± 0.78 nM) was more potent than the Z‐isomer (IC50: 52.6 ± 0.3 nM) and displayed a relatively high dopamine:serotonin transporter selectivity (28‐fold). In radiolabeled form, [125I]altropane bound to sites in the striatum with a single high affinity (KD: 5.33 ± 0.55 nM) and with a site density (BMAX: 301 pmol/g original wet tissue weight) that was within the density range reported previously for the dopamine transporter in striatum. Drugs inhibited [125I]altropane binding with a rank order of potency that corresponded closely to their potencies for inhibiting [3H]WIN 35,428 binding (r2: 0.99; P < 0.0001) to the blocking dopamine transport. The favorable binding properties of altropane, together with its rapid entry into primate brain and highly localized distribution in dopamine‐rich brain regions, suggest it is a suitable iodinated probe for monitoring the dopamine transporter in vitro and in vivo by SPECT or PET imaging. Synapse 29:93–104, 1998.

Bicyclo[3.2.1]octanes: Synthesis and inhibition of binding at the dopamine and serotonin transporters

Herein we report the synthesis of a series of bicyclo[3.2.1]octanes and their binding characteristics at the dopamine and serotonin transporters. The data confirm that a heteroatom at position 8 of the tropane nucleus is not a prerequisite for binding since the bicyclo[3.2.1]octanes prove potent inhibitors of both transporters. Therefore the three-dimensional topology of the ligand may be more important than specific functionality with respect to stereospecific binding at the acceptor site.

Dopamine Transporter (DAT) Inhibitors Alleviate Specific Parkinsonian Deficits in Monkeys: Association with DAT Occupancy in Vivo

Viable dopamine neurons in Parkinsons disease express the dopamine transporter (DAT) and release dopamine (DA). We postulated that potent DAT inhibitors, with low affinity for the serotonin transporter (SERT), may elevate endogenously released extracellular dopamine levels to provide therapeutic benefit. The therapeutic potential of eight DAT inhibitors was investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated cynomolgus monkeys (Macaca fascicularis), with efficacy correlated with DAT occupancy as determined by positron emission tomography imaging in striatum. Four potent DAT inhibitors, with relatively high norepinephrine transporter, but low SERT affinities, that occupied the DAT improved activity in parkinsonian monkeys, whereas three high-affinity DAT inhibitors with low DAT occupancy did not. 2β-Carbomethoxy-3α-(3,4-dichlorophenyl)-7β-hydroxy-8-methyl-8-azabicyclo[3.2.1.]octane (O-1163) occupied the DAT but had short-lived pharmacological effects. The benztropine analog difluoropine increased general activity, improved posture, reduced body freeze, and produced sleep disturbances at high doses. (1R)-2β-(1-Propanoyl)-3α-(4-fluorophenyl)tropane (O-1369) alleviated parkinsonian signs in advanced parkinsonian monkeys, by increasing general activity, improving posture, reducing body freeze, and sedation, but not significantly reducing bradykinesia or increasing locomotor activity. In comparison with the D2-D3 DA receptor agonist quinelorane, O-1369 elicited oral/facial dyskinesias, whereas quinelorane did not improve posture or reduce balance and promoted stereotypy. In conclusion, DAT inhibitors with therapeutic potential combine high DAT affinity in vitro and high DAT occupancy of brain striatum in vivo with enduring day-time effects that do not extend into the nighttime. Advanced parkinsonian monkeys (80% DAT loss) respond more effectively to DAT inhibitors than mild parkinsonian monkeys (46% DAT loss). The therapeutic potential of dopamine transport inhibitors for Parkinsons disease warrants preclinical investigation.