A non-anhydrous, minimally basic protocol for the simplification of nucleophilic 18F-fluorination chemistry.

Fluorine-18 radiolabeling typically includes several conserved steps including elution of the [18F]fluoride from an anion exchange cartridge with a basic solution of K2CO3 or KHCO3 and Kryptofix 2.2.2. in mixture of acetonitrile and water followed by rigorous azeotropic drying to remove the water. In this work we describe an alternative "non-anhydrous, minimally basic" ("NAMB") technique that simplifies the process and avoids the basic conditions that can sometimes limit the scope and efficiency of [18F]fluoride incorporation chemistry. In this approach, [18F]F- is eluted from small (10-12 mg) anion-exchange cartridges with solutions of tetraethylammonium bicarbonate, perchlorate or tosylate in polar aprotic solvents containing 10-50% water. After dilution with additional aprotic solvent, these solutions are used directly in nucleophilic aromatic and aliphatic 18F-fluorination reactions, obviating the need for azeotropic drying. Perchlorate and tosylate are minimally basic anions that are nevertheless suitable for removal of [18F]F- from the anion-exchange cartridge. As proof-of-principle, "NAMB" chemistry was utilized for the synthesis of the dopamine D2/D3 antagonist [18F]fallypride.

Antidepressant-like effects of the novel kappa opioid antagonist MCL-144B in the forced-swim test.

Previous studies have demonstrated that kappa opioid receptor (KOR) antagonists reduce stress- and depression-like behaviors. We hypothesized that administration of a novel opioid mixed agonist/antagonist capable of antagonist activity at the KOR would attenuate forced-swim stress (FSS)-induced immobility, an animal model of depression-like behavior. C57Bl/6J mice were exposed to antinociceptive and repeated FSS testing after pretreatment with a graded dose of a novel bivalent morphinan compound, bis(N-cyclobutylmethylmorphinan-3-yl) sebacoylate dihydrochloride (MCL-144B). MCL-144B demonstrated dose- and time-dependent antinociception and KOR-mediated antagonism. In support of the hypothesis, pretreatment with MCL-144B dose-dependently attenuated stress-induced antinociception and immobility in the forced-swim test.

Synthesis and radiopharmacological characterization of 2beta-carbo-2'-[18F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane ([18F]MCL-322) as a PET radiotracer for imaging the dopamine transporter (DAT).

The fluoroalkyl-containing tropane derivative 2beta-carbo-2'-fluoroethoxy-3beta-(4-bromo-phenyl)tropane (MCL-322) is a highly potent and moderately selective ligand for the dopamine transporter (DAT). The compound was labeled with the short-lived positron emitter (18)F in a single step by nucleophilic displacement of the corresponding tosylate precursor MCL-323 with no-carrier-added [(18)F]fluoride. The positron emission tomography (PET) radiotracer 2beta-carbo-2'-[(18)F]fluoroethoxy-3beta-(4-bromo-phenyl)tropane [(18)F]MCL-322 was obtained in decay-corrected radiochemical yields of 30-40% at a specific radioactivity of 1.6-2.4Ci/mumol (60-90GBq/mumol) at the end-of-synthesis (EOS). Small animal PET, ex vivo and in vivo biodistribution experiments in rats demonstrated a high uptake in the striatum (3.2% ID/g) 5min after injection, which increased to 4.2% ID/g after 60min. The uptake in the cerebellum was 1.8% ID/g and 0.6% ID/g after 5min and 60min post-injection, respectively. Specific binding to DAT of [(18)F]MCL-322 was confirmed by blocking experiments using the high affinity DAT ligand GBR 12909. The radiopharmacological characterization was completed with metabolite and autoradiographic studies confirming the selective uptake of [(18)F]MCL-322 in the striatum. It is concluded that the simple single-step radiosynthesis of [(18)F]MCL-322 and the promising radiopharmacological data make [(18)F]MCL-322 an attractive candidate for the further development of a PET radiotracer potentially suitable for clinical DAT imaging in the human brain.

Synthesis and biological evaluation of a series of novel N- or O-fluoroalkyl derivatives of tropane: potential positron emission tomography (PET) imaging agents for the dopamine transporter.

A series of novel fluoroalkyl-containing tropane derivatives was synthesized, and their binding affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) were determined via competitive binding assays. Among these derivatives, the fluoropropyl ester of beta-CIT (19), the fluoroethyl ester of beta-CIT (20), the N-fluoropropyl derivative of beta-CBT (12), and the fluoropropyl ester of beta-CMT (18) displayed higher affinity and greater selectivity for the DAT versus SERT and NET than FP-CIT, which indicates that they are attractive candidates for the development of (18)F-labeled PET imaging agents for the DAT.

Mixed kappa agonists and mu agonists/antagonists as potential pharmacotherapeutics for cocaine abuse: synthesis and opioid receptor binding affinity of N-substituted derivatives of morphinan.

A series of new N-substituted derivatives of morphinan was synthesized and their binding affinity for the three opioid receptors (mu, delta, and kappa) was determined. A paradoxical effect of N-propargyl (MCL-117) and N-(3-iodoprop-(2E)-enyl) (MCL-118) substituents on the binding affinities for the mu and kappa opioid receptors was observed. All of these novel derivatives showed a preference for the mu and kappa versus delta binding.

Kappa opioid agonists as targets for pharmacotherapies in cocaine abuse.

Kappa opioid receptors derive their name from the prototype benzomorphan, ketocyclazocine (1a) which was found to produce behavioral effects that were distinct from the behavioral effects of morphine but that were antagonized by the opioid antagonist, naltrexone. Recent evidence suggests that agonists and antagonists at kappa opioid receptors may modulate the activity of dopaminergic neurons and alter the neurochemical and behavioral effects of cocaine. Kappa agonists blocked the effects of cocaine in squirrel monkeys in studies of cocaine discrimination and scheduled-controlled responding. Studies in rhesus monkeys suggested that kappa opioids may antagonize the reinforcing effects of cocaine. These studies prompted the synthesis and evaluation of a series of kappa agonists related to the morphinan, L-cyclorphan (3a) and the benzomorphan, L-cyclazocine (2). We describe the synthesis and preliminary evaluation of a series of morphinans, structural analogs of cyclorphan 3a-c, the 10-keto morphinans 4a and b, and the 8-keto benzomorphan 1b, structurally related to ketocyclazocine (1a). In binding experiments L-cyclorphan (3a), the cyclobutyl (3b), the tetrahydrofurfuryl 3c and the 10-keto 4b analogs had high affinity for mu (mu), delta (delta) and kappa (kappa) opioid receptors. Both 3a and 3b were more selective for the kappa receptor than the mu receptor. However, 3b was 18-fold more selective for the kappa receptor in comparison to the delta receptor, while cyclorphan (3a) had only a 4-fold greater affinity for the kappa receptor in comparison to the delta receptor. The cyclobutyl compound 3b was found to have significant mu agonist properties, while 3a was a mu antagonist. All compounds were also examined in the mouse tail flick and writhing assay. Compounds 3a and 3b were kappa agonists. Correlating with the binding results, compound 3a had some delta agonist properties, while 3b was devoid of any activity at the delta receptor. In addition, compounds 3a and 3b had opposing properties at the mu opioid receptor. The cyclobutyl compound 3b was found to have significant mu agonist properties, while 3a was a mu antagonist.

[123I]IPCIT and [123I]beta-CIT as SPECT tracers for the dopamine transporter: a comparative analysis in nonhuman primates.

[123I]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([123I]-CIT) and its isopropylester analog [123I]PCIT, both of which are phenyltropane derivatives of cocaine with high affinity for the dopamine (DA) transporter, were compared using single photon emission computed tomography in nonhuman primates. Although IPCIT is significantly more selective for the DA transporter than beta-CIT, striatal distribution volumes of specifically bound tracer were similar for both tracers. Compartmental modeling results were compared with a simple peak equilibrium method used previously by this group. The peak equilibrium method is shown to overestimate striatal distribution volumes, primarily due to a difference in the calculated time of peak specific uptake.

Synthesis and opioid receptor affinity of morphinan and benzomorphan derivatives: mixed kappa agonists and mu agonists/antagonists as potential pharmacotherapeutics for cocaine dependence.

This report concerns the synthesis and preliminary pharmacological evaluation of a novel series of kappa agonists related to the morphinan (-)-cyclorphan (3a) and the benzomorphan (-)-cyclazocine (2) as potential agents for the pharmacotherapy of cocaine abuse. Recent evidence suggests that agonists acting at kappa opioid receptors may modulate the activity of dopaminergic neurons and alter the neurochemical and behavioral effects of cocaine. We describe the synthesis and chemical characterization of a series of morphinans 3a-c, structural analogues of cyclorphan [(-)-3-hydroxy-N-cyclopropylmethylmorphinan S(+)-mandelate, 3a], the 10-ketomorphinans 4a,b, and the 8-ketobenzomorphan 1b. Binding experiments demonstrated that the cyclobutyl analogue 3b [(-)-3-hydroxy-N-cyclobutylmethylmorphinan S(+)-mandelate, 3b, MCL-101] of cyclorphan (3a) had a high affinity for mu, delta, and kappa opioid receptors in guinea pig brain membranes. Both 3a,b were approximately 2-fold more selective for the kappa receptor than for the mu receptor. However 3b (the cyclobutyl analogue) was 18-fold more selective for the kappa receptor in comparison to the delta receptor, while cyclorphan (3a) had only 4-fold greater affinity for the kappa receptor in comparison to the delta receptor. These findings were confirmed in the antinociceptive tests (tail-flick and acetic acid writhing) in mice, which demonstrated that cyclorphan (3a) produced antinociception that was mediated by the delta receptor while 3b did not produce agonist or antagonist effects at the delta receptor. Both 3a,b had comparable kappa agonist properties. 3a,b had opposing effects at the mu receptor: 3b was a mu agonist whereas 3a was a mu antagonist.

[3H]beta-CIT: a radioligand for dopamine transporters in rat brain tissue.

[3H]2-beta-carbomethoxy-3-beta-[4'-iodophenyl]tropane (beta-CIT) was prepared and evaluated. With rat forebrain tissue, [3H]beta-CIT showed high affinity for dopamine transporters (DAT), with selectivity for DAT over norepinephrine transporters, but not serotonin transporters, as well as DAT-stereoselectivity with beta-CIT, amphetamine and methylphenidate. Affinity and selectivity for 53 compounds assayed with [3H]beta-CIT and standard DAT radioligand [3H]GBR-12935 were highly correlated (r0.95). [3H]beta-CIT is proposed as a useful, high-affinity DAT radioprobe.

Selective alkylatation of dopamine D2 and D4 receptors in rat brain by N-(p-isothiocyanatophenethyl)spiperone.

Effects of the D2-like receptor alkylating agent NIPS (N-[p-isothiocyanatophenethyl]spiperone) on dopamine receptors in rat brain were characterized by radioreceptor assays and quantitative autoradiography. NIPS alkylated D2 and D4 receptors concentration-dependently in brain sections and transfected cells. NIPS also alkylated both receptors dose-dependently in vivo, with no effect on dopamine D1-like or serotonin 5-HT2 receptors at a dose that occluded 75% of D2 and D4 receptors. Pretreatment with D2-like receptor selective antagonist haloperidol completely blocked the effects of NIPS. The findings demonstrate that NIPS selectively alkylates D2 and D4 receptors, indicating its potential utility for studies of these receptors.

Medial prefrontal cortical D2 and striatolimbic D4 dopamine receptors: common targets for typical and atypical antipsychotic drugs.

1. In vitro receptor autoradiography was used to examine the long-term effects of a typical (fluphenazine), atypical (clozapine), or potential atypical antipsychotic (S[+]-N-n-propylnorapomorphine; [+]-NPA) on different dopamine (DA) receptor subtypes. 2. D1-Like and D3 receptor levels were not changed with any treatment in any brain region examined. 3. D2 Receptors in caudate-putamen (CPu), nucleus accumbens (NAc) and olfactory tubercle (OT) were significantly increased by long-term treatment with fluphenazine, but not with clozapine or S[+]-NPA. 4. D2 Receptor levels in medial prefrontal cortex (MPC), but not dorsolateral frontal cortex (DFC), were elevated after repeated daily administration of fluphenazine, clozapine, and S[+]-NPA. 5. D4-Like receptors, assayed under D4-selective conditions, were increased by fluphenazine, clozapine and S(+)-NPA in both NAc and CPu, but by none of these treatments in OT, DFC or MPC. 6. These results support a common role for medial prefrontal cortical D2 and striatolimbic D4 receptors in mediating the clinical actions of typical and atypical antipsychotic drugs.

Local injection of alkylating and nonalkylating dopamine receptor antagonists into rat basal forebrain: autoradiographic assessment of D2-like and D3 sites.

N-chloroethyl derivatives of 7-hydroxy-1,2,3,4-tetrahydronaphthalene (7-OH-DPAT), 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), or fluphenazine were microinjected into rat nucleus accumbens (Acc), and receptor binding quantified autoradiographically after 24 h. EEDQ reduced [3H]nemonapride (D2-like receptors) binding in Acc (by 84%) and islands of Calleja (IC; 44%), without affecting [3H](+)-7-OH-DPAT (D3); N-chloroethyl-7-OH-DPATs blocked both radioligands in Acc and IC (30%-70%); fluphenazine had no effect.

Long-term effects of S(+)N-n-propylnorapomorphine compared with typical and atypical antipsychotics: differential increases of cerebrocortical D2-like and striatolimbic D4-like dopamine receptors.

Changes in D2-like dopamine (DA) receptor binding in rat brain regions were compared by quantitative in vitro receptor autoradiography after 21-d treatment with a typical (fluphenazine), atypical (clozapine), or candidate atypical antipsychotic (S[+]-N-n-propylnorapomorphine, [+]-NPA). Fluphenazine treatment significantly increased binding of the D2,3,4 radioligands [3H]nemonapride and [3H]spiperone in caudate-putamen (CPu: 22%, 32%), nucleus accumbens (ACC: 67%, 52%), olfactory tubercle (OT: 53%, 43%), and medial prefrontal cerebral cortex (MPC: 46%, 47%) but not dorsolateral frontal cortex (DFC). D2-like binding in MPC was also increased by (+)-NPA (49%, 39%) and clozapine (60%, 40%), but not in DFC, CPu, ACC, or OT. Binding of D2,3-selective [3H]raclopride increased less after fluphenazine in ACC (27%) and CPu (16%) than with the nonselective radioligands, and not after clozapine or (+)-NPA. D3-selective binding of [3H]R (+)-7-OH-DPAT was not changed with any treatment or region including islands of Calleja. Binding of [3H]nemonapride or [3H]spiperone under D4-selective conditions (with 300 nM S[-]-raclopride and other masking agents, at sites occluded by D4 ligand L-745,870), was increased by fluphenazine, (+)-NPA, clozapine in ACC (120%, 76%, 70%, respectively), and CPu (54%, 37%, 35%), but not in OT, DFC or MPC. These results support the hypothesis that cerebrocortical D2-like and striatolimbic D4-like receptors contribute to antipsychotic actions of both typical and atypical drugs and encourage further consideration of S(+)aporphines as potential atypical antipsychotics.

Effect of the haloperidol tetrahydropyridine metabolite 4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3,6- tetrahydropyridine on dopamine receptor and transporter binding. A nonhuman primate 123I-iodobenzamide and 2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane single photon emission computed tomographic study.

Researching the biological activities and toxicities of metabolites of drugs is of growing importance and has received increasing attention during the last decade in order to gain a better understanding of the efficacy and safety profile of drugs in clinical use. HPTP (4-(4-chlorophenyl)-1-[4-(4-fluorophenyl)-4-oxobutyl]-1,2,3, 6-tetrahydropyridine, CAS 52669-92-8), the tetrahydropyridine metabolite of the classical neuroleptic, haloperidol (CAS 52-86-8), has recently been the focus for further understanding the well-known side effect profile of haloperidol. The current study was aimed at investigating the effect of HPTP treatment on dopamine receptor and transporter binding in the nonhuman primate, i.e. the baboon Papio ursinus. The study was performed using the dopamine receptor ligand, 123-I-iodobenzamide (IBZM) and the dopamine transporter ligand, [123]2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane (beta-CIT) in planar scintigraphy and single photon emission computed tomographic (SPECT) protocols. Dopamine receptor binding in the striatum was measured from the time activity curves by calculating the IBZM ratios of the basal ganglia to frontal cortex and of the basal ganglia to cerebellum. 99mTc-HMPAO (hexamethylpropylene amine oxime) SPECT detected no changes in striatal perfusion during HPTP treatment. The transporter binding was measured by dynamic imaging of the basal ganglia, frontal cortex and cerebellum using beta-CIT. IBZM dopamine receptor binding is initially (as measured after 18 weeks treatment) decreased by HPTP treatment in the basal ganglia, frontal cortex (not significantly) and cerebellum but reversed to control values in the frontal cortex, as measured after 58 weeks treatment with HPTP. The binding to the basal ganglia and to a lesser degree the cerebellum is still affected after 58 weeks treatment with HPTP but indicates a tendency to return towards the control values. The results of the planar dynamic study with beta-CIT indicate a decrease in the beta-CIT binding to the dopamine transporters in the basal ganglia and to a lesser extent the cerebellum as measured by the time activity and percentage washout rate of the beta-CIT in the HPTP treated baboons. The effect of HPTP on the serotonin transporters appears to be minimal as observed from the results obtained from the frontal cortex. These results indicate that HPTP treatment influences both presynaptic and postsynaptic neurofunction in the dopaminergic neurones.

Human biodistribution and dosimetry of iodine-123-fluoroalkyl analogs of beta-CIT.

Two new N-omega-fluoroalkyl analogs of [123I]2beta-carbomethoxy-3beta-(4-iodophenyl)tropane ([123I]beta-CIT), the fluoroethyl and fluoropropyl compounds ([123I]FE-CIT and [123I]FP-CIT, respectively), have been shown to have faster kinetics and better selectivity for the dopamine transporter than [123I]beta-CIT. We examined the organ biodistribution and radiation safety of these two compounds in six healthy volunteers who received an injection with each of the two compounds 2 weeks apart. Data were obtained on the Strichman 860 whole-body scanner. Transmission scans were obtained in all subjects prior to the injection of the radiotracer with a line source and used to derive organ-specific attenuation correction factors. Whole-body planar images were acquired every hour for the first 6 h, and at 24 h. Attenuation-corrected regional conjugate counts were converted into units of activity using a calibration factor obtained for each subject by dividing whole-body conjugate decay-corrected counts from the first acquisition by the injected activity. Radiation dose estimates were on average higher for [123I]CIT-FE than for [123I]CIT-FP, with the lower large intestine receiving the highest exposure: 0.15+/-13% mGy/MBq (mean +/-COV) and 0.12+/-14% mGy/MBq for [123I]FE-CIT and [123I]FP-CIT, respectively, followed by the upper large intestine and the spleen.

Radiosynthesis of [18F] N-3-fluoropropyl-2-beta-carbomethoxy-3-beta-(4-iodophenyl) nortropane and the first human study with positron emission tomography.

A procedure for the routine preparation of [18F]FP-CIT has been developed. Purification of the final product was achieved by preparative HPLC using phenethyl column without decomposition or epimerization. [18F] labeled-N-fluoropropyl-2 beta-carbomethoxy-3 beta-(4-iodophenyl) nortropane was prepared and PET imaging was performed on human subjects. A high uptake into striatal regions was observed. HPLC plasma analysis using [18F]FP-CIT indicated the presence of only one metabolite. By directly comparing the behavior of these three radiotracers ([18F]DOPA, [123I]FP-CIT, and [18F]FP-CIT) in the same subjects, we can enhance our understanding of the dopaminergic system as well as the relative potential of these techniques in a clinical research setting.

SPECT imaging of dopamine transporters in human brain with iodine-123-fluoroalkyl analogs of beta-CIT.

Iodine-123-2 beta-carbomethoxy-3 beta-(4-iodophenyl)tropane (beta-CIT) is a useful SPECT tracer for imaging the dopamine transporter. Its slow kinetics, however, necessitate imaging on the day after the injection. Two N-omega-fluoroalkyl analogs of beta-CIT, the fluoropropyl and fluoroethyl compounds (beta-CIT-FP and beta-CIT-FE, respectively), characterized by faster kinetics in baboons, were tested in humans as potential tracers for the dopamine transporter. Four healthy volunteers were injected with [123I]-beta-CIT-FP and another four were injected with [123I]beta-CIT-FE. SPECT data were acquired for 1149 +/- 590 min and 240 +/- 30 min, respectively. Both tracers demonstrated high brain uptake (6.37% +/- 0.37% and 7.8% +/- 1.5% of the injected dose, respectively). Activity concentrated with time in the striatal area, reaching a peak within 30 min, with little or no washout for [123I]beta-CIT-FP and a faster washout for [123I]beta-CIT-FE (14.7% +/- 6.9%). Occipital and midbrain activity showed similar patterns, displaying a peak within 15 min and rapid washout, followed by stable levels at approximately 100 min for both tracers. The ratio of peak specific striatal-to-peak specific midbrain activity was 9.1 +/- 1.8 for [123I]beta-CIT-FP and 7.7 +/- 0.7 for [123I]beta-CIT-FE, showing high in vivo selectivity for the dopamine transporter. These preliminary results suggest that both compounds could be used as SPECT (labeled with 123I) or PET (labeled with 18F) radiotracers to image the dopamine transporters in the living human brain.

Improved models for pharmacological null experiments: calculation of drug efficacy at recombinant D1A dopamine receptors stably expressed in clonal cell lines.

Modern drug discovery demands accurate knowledge of the drug properties of affinity and efficacy at specific receptor proteins. Furthermore, drugs with well defined properties make better tools with which to explore and understand receptor regulation. The use of clonal cell lines stably expressing a given recombinant receptor may provide a highly useful model in which drug effects may be studied on one receptor subtype at a time. The present report was designed to evaluate the utility of a general method in which a clonal cell line stably expressing a recombinant D1A dopamine receptor was used as a model system for studying drug actions by null models. The null model for receptor occlusion (to calculate agonist Ka) and the null model for relative efficacy (to calculate test agonist affinity and epsilon r) were evaluated in these studies. To initiate these studies, rat C6 glioma cells that do not normally express DA receptors have been modified by stable transfection with the primate D1A DA receptor [Machida et al., 1992 (Molec. Pharmacol. 41: 652-659)] to a density of approximately equal to fmol/mg protein. The recombinant receptors show robust stimulation of cAMP in the stably transfected C6 cells. Calculation of agonist Ka from dose-response data requires that a portion of the cell's receptors be occluded in the absence of changes in post-receptor events leading to the response. Receptor reserve is typically reduced by alkylation, thereby lowering maximal response. Unfortunately, most of the currently available alkylating agents are not selective either for a particular receptor or for receptors vs other proteins within a signaling pathway. Short-term agonist treatment offers a possible complement to the use of non-selective or poorly characterized alkylating drugs for reducing maximum response in appropriate cell systems. The null method of receptor occlusion was used to determine the Ka for dopamine when maximum response was decreased by alkylation vs short-term agonist treatment. Direct non-linear curve fitting was used to analyze the data. In addition to DA, two other compounds were used to reduce receptor reserve to validate the method: fenoldopam (relatively high efficacy) and SKF38393 (low efficacy). Analyses indicated that the affinity of DA was similar whether calculated by alkylation (1.1 +/- 0.58 microM), 75 min DA treatment (0.57 +/- 0.16 microM) or 45 min treatment with DA (0.86 +/- 0.11 microM). Short-term agonist treatment experiments using multiple concentrations of DA, fenoldopam, or SKF38393 to decrease receptor reserve provided additional support for the validity of the Ka determinations using this procedure. Other experiments were conducted according to the null model for relative efficacy in which the affinity for DA is calculated by comparing the DA response before and after receptor occlusion, and the affinity and relative intrinsic efficacy of the test agonist are determined as a function of its actions relative to DA. We used the following four test drugs: + Br-APB, a novel agent with potential dopamine agonist properties, and three high-affinity DA agonists, fenoldopam, R-(-)-apomorphine (APO), and SKF38393. Intrinsic efficacy values relative to that of DA (1.0) were as follows: fenoldopam, 0.46 +/- 0.11; APO, 0.19 +/- 0.13; SKF38393, 0.07 +/- 0.01; and +Br-APB, 0.26 +/- 0.40. The agonist affinities (Ka) were: fenoldopam, 0.018 +/- 0.008 microM; APO, 0.80 +/- 0.18 microM; SKF38393, 0.16 +/- 0.04 microM; BR-APB, 0.43 +/- 0.29 microM; and DA, 0.58 +/- 0.17 microM. EC50/Ka ratios were consistent with relative intrinsic efficacies and Ka values were similar to KL values reported for membrane binding studies. Finally, Monte Carlo simulations were conducted to determine the precision of the parameter estimates...

Reproducibility of iodine-123-beta-CIT SPECT brain measurement of dopamine transporters.

UNLABELLED: Iodine-123-beta-CIT has been used as a probe of monoamine transporters in human and nonhuman primates utilizing SPECT. To assess the utility of this tracer for measurement of striatal dopamine (DA) transporters in human disease, we studied the test/retest variability and reliability of SPECT measures obtained after bolus injection of [123I]beta-CIT 0-7 hr (Day 1) and 18-24 hr (Day 2) after administration. METHODS: For the Day 2 study, seven healthy humans (4 men, 3 women; aged 19-74 yr) participated in two [123I]beta-CIT SPECT scans separated by 7-14 days. Subjects were imaged at 18, 21 and 24 hr postinjection of 370 MBq (10 mCi) [123I]beta-CIT. Two outcome measures were evaluated: (a) the ratio of specific striatal (activity associated with DA transporter binding) to nondisplaceable uptake, also designated V"3 and (b) the total, specific striatal uptake (%SSU) expressed as a percentage of injected radiotracer dose. Test/retest variability associated with V"3 and total specific striatal uptakes were compared for scans acquired at 18, 21 and 24 hr with 24 hr only postinjection scans. For the Day 1 study, three of the subjects participated in two kinetic studies of [123I]beta-CIT uptake. A three-compartment model was used for determination of konBmax and binding potential (BP = Bmax/Kd) and the reproducibility of the measures assessed. RESULTS: In the Day 2 study, both outcome measures demonstrated excellent test/retest reproducibility with variability of V"3 = 6.8 +/- 6.8% and percent striatal uptake = 6.6 +/- 4.3% using data acquired from all time points. There were no significant differences in variability for the two outcome measures obtained. The intraclass correlation coefficient rho was 0.96 and 0.98 for V"3 and %SSU, respectively. Considering the 24 hr postinjection scans only, there was a nonsignificant trend toward lower test/retest variability for %SSU compared to V"3 (6.6 +/- 4.2% and 12.8 +/- 9.0%, respectively). The test/retest variability for the Day 1 kinetic modeling data showed marked differences depending on the fitting strategy and assumptions about the reversibility of [123I]beta-CIT in striatum. Using a model that assumed a low, fixed value for reversible striatal binding (k4) produced low variability (12 +/- 9%). CONCLUSION: These data suggest that SPECT imaging performed at either 0-7 hr or 18-24 hr after [123I]beta-CIT injection permits calculation of reliable and reproducible measures of dopamine transporters and supports the feasibility of using [123I]beta-CIT in serial evaluation of human neuropsychiatric disease.